JP7000321B2 - 5-Hydroxytryptamine 1B receptor stimulant for use as a promoter of self-renewal and / or differentiation of satellite cells - Google Patents

Description

Introduction The present invention relates to the field of muscle regeneration, more particularly to the replacement of in vivo muscle stem cell pools. More specifically, the present invention is 5) for use as an agent for promoting self-renewal and / or differentiation of satellite cells, and / or ii) an agent for preventing and / or inhibiting the depletion of a satellite cell pool. -Regarding an agent that directly or indirectly stimulates the hydroxytryptamine 1B receptor, and a composition comprising the agent. The present invention further includes therapeutic and screening methods.

Satellite cells are muscle stem cells that are located between muscle fibers and their basement membranes and are involved in the development and regeneration of muscle tissue.

In mature muscle, satellite cells are generally quiescent, but can be recruited as needed after minor or massive muscle trauma. With minimal muscle damage, satellite cells and / or their progeny fuse with existing muscle fibers, whereas during large-scale muscle damage, satellite cells fuse with each other to create new muscle fibers. (Grounds and Yablonka-Reuveni, 1993; Hawke and Garry, 2001). However, since fine muscle fiber damage occurs routinely during normal muscle activity, continuous repair requirements are essential for muscle maintenance. This repair is probably due to the sustained self-renewal of satellite cells, which involves a complex process that involves not only the proliferation of these cells, but also their storage and differentiation into mature muscle cells.

Myosatellite proliferation is generally activated during mild or severe damage or destruction of the basement membrane, which eventually leads to the differentiation of some of these cells into new myogenic cells. , Others lead to the re-establishment of a surplus in vivo reserve of quiescent cells capable of supporting the next regeneration (Moss and Leblond, 1971; Schulz and Jaryszak, 1985; Bischoff, 1994). This "two-fate" process, more specifically, follows the following steps that can be followed by monitoring the transcriptome and molecular profile of the biomarkers expressed in these cells: In regenerating muscle, satellite cell pools contain heterozygous Pax7 + cells that are most notably identified based on Myf5 expression at some point in their lineage. These Pax7 + / Myf5-cells (ie, cells that have never expressed Myf5 and have never had a progenitor cell that expressed Myf5) have the ability to self-renew and differentiate into Pax7 + / Myf5 + cells. Corresponds to a certain group of. Once Myf5 expression is found intracellularly, the cells and their progeny are destined for proliferation and differentiation (Kuang, Kuroda et al. 2007). These differences are achieved through asymmetric cell division, which is governed by the physical properties of the satellite cell pool. Each satellite cell actually has a basal side in contact with the basement membrane and an apical side in contact with the host muscle fiber. When satellite cells divide, daughter cells adjacent to the basement membrane undergo self-renewal, while daughter cells in contact with muscle fibers undergo temporary amplification and differentiation. As this muscle-guided pathway progresses, MyoD will also be expressed in Pax7 + / Myf5 + activated cells (ie, self-regenerating cells). However, again, when the satellite cells are subjected to differentiation, there is a population of self-regenerating MyoD-cells that express Myf5, and these cells can actually dedifferentiate and replenish the satellite cell niche. It has been observed (Baroffio, Hamman et al., 1996; Beauchamp, Helsop et al., 2000). In differentiated cells, with sustained expression of MyoD and Myf5, expression of myogenin and MRF4 (even among others) is upregulated, but expression of Pax7 is reduced (Smith, Janney). et al., 1994; Yablonka-Reuveni, 1994; Cornelison and Wold, 1997). This results in cell cycle quiescence mediated by p21 activation and expression of muscle-specific proteins such as myosin heavy chains (Charge and Rudnicki, 2004), followed by M-cadherin for forming mature polynuclear myofibers, Upregulation of expression of intermediate filament proteins such as m-caplain, as well as desmin and vimentin (Kuch et al .; 1997; Kwak et al., 1993; Smythe et al, 2001; Vaittinen et al., 2001) is brought about. Thus, muscle regeneration is a complex multi-step process evoked by satellite cell activation that drives both self-renewal of satellite cells and differentiation into mature muscle fibers.

That said, the ability of satellite cells to regenerate is not infinite. In fact, age-related declines in satellite cell abundance and / or function further limit muscle fiber repair and contribute to age-related muscle loss (Bentzinger et al., 2014; Cosgrove et al.). , 2014; Bern et al., 2014; Goodell et al., 2015). Age-related muscle disorders such as sarcopenia have been reported in both humans and animals and are characterized by decreased skeletal muscle mass, strength and endurance, which increase the susceptibility to contraction-induced muscle damage. I can invite you. It has also been confirmed that depletion of the satellite cell population is an important factor in the exacerbation and death of patients affected by congenital muscular disorders such as Duchenne muscular dystrophy. Kudryashova et al. (2012) report, in particular, that aging of satellite cells is a fundamental feature of myopathy in the rodent model of muscular dystrophy. However, the exact involvement of satellite cells in congenital and acquired muscle disorders is not fully understood.

Therefore, whether the reduction of the in vivo pool of satellite cells results from natural (age-related) loss and / or damage and / or damage to skeletal muscle tissue, pathological (congenital or damage-related) loss and There is a continuous demand for functional satellite cells throughout life, whether resulting from / or injury and / or disability.

The present invention addresses the above requirements in the art.

In particular, we have surprisingly and unexpectedly found that fluoxetine and vortioxetine promote the proliferation of satellite cells, especially by increasing their rate of division. They increase the diameter of muscle fibers, especially in the in vivo Duchenne muscular dystrophy model, and promote muscle regeneration in a sustainable manner even after multiple injuries. Thus, the results show that these antidepressants can be used to induce muscle regeneration and slow the progression of muscular dystrophy. We also found that these amazing effects were mediated by the 5-hydroxytryptamine 1B receptor expressed in muscle tissue, which directly or indirectly mediated the 5-hydroxytryptamine 1B receptor. It is suggested that any (selective or non-selective) drug that stimulates the satellite cells will exert a similar beneficial effect on satellite cells.

Therefore, the present invention
i) 5-Hydroxytryptamine 1B receptor stimulator for use as an agent to promote self-renewal and / or differentiation of satellite cells; and / or ii) to prevent and / or inhibit the depletion of satellite cell pools, And the composition comprising the said agent.

The invention further includes in vitro use, as well as therapeutic and screening methods.

Specific description of the invention

Unless indicated otherwise, the scientific and technical terms used with respect to the present invention shall have meanings commonly understood by those of skill in the art. Moreover, the nomenclature and molecular biology, cell culture, and pharmacological techniques used herein are well known and commonly used in the art, unless the context requires otherwise. .. Such techniques are well documented in the literature (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 2013; Remington: The Science and Practice of Pharmacy, 22nd Edition. , Mack Publishing Co., Easton, Pa., 2012).

However, with respect to the use of various terms herein, the following definitions apply in more detail.

With respect to the present invention, the terms "5-hydroxytryptamine receptor", "5-HT receptor", "5-HT R" or "serotonin receptor" are found in almost all animal central and peripheral nervous systems. Means a superfamily of single polypeptide 7-transmembrane receptors that act through activation of the G protein signaling pathway and / or as a ligand-gated ion channel. The 5-hydroxytryptamine receptor is a neurotransmitter such as glutamate, GABA, dopamine, epinephrine / norepinephrine, and acetylcholine, and above all, many hormones such as oxytocin, prolactin, vasopresin, cortisol, corticotropin, and substance P. It is activated by their natural ligand, serotonin, to regulate release.

5-Hydroxytryptamine receptors are further categorized into 7 groups according to their G protein conjugation, of which the 5-HT1 group is particularly targeted for the present invention.

The "5-HT1 receptor group" comprises the 5-HT1 A, B, D, E and F subtypes, having structural homology between 40-63% in humans and the Gαi / o protein. Meet with priority. Activation of 5-HT1 receptor subtypes generally induces inhibitory neurotransmission through activation of potassium channels and reduces intracellular cAMP production.

Among the 5-HT1 receptor groups, "5-HT1 B receptor"("5-hydroxytryptamine 1B receptor", "5-hydroxytryptamine receptor 1B", "5-HT-1B", "5-""HT-1D-β","serotonin 1Dβ receptor", or "serotonin receptor 1B") was identified and characterized by Jin et al. In 1992. In humans, this receptor is encoded by the HTB1R gene (NCBI RefSeq consignment version numbers NM_000863.1 and GI: 4504532; corresponding coding proteins: NCBI RefSeq consignment version number NP_000854.1 and GI: 45045533) and chromosome 6. It is located at 6q13 of. The sequence of this receptor is human, chimpanzee, rhesus monkey, dog, bovine, mouse, rat, chicken, zebrafish, C.I. Well conserved in C. elegans , and frogs, it is known that 135 organisms have homologous molecular species with the human gene HTR1B.

The "5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant" or "5-HT1 BR stimulant", as used herein, directly or indirectly determines the activity of the 5-hydroxytryptamine 1B receptor. It means an activator that can be stimulated, or in other words, that can mimic or enhance the effects normally exerted by the natural ligand of the receptor (ie, serotonin). Direct stimuli require the binding of the drug to the receptor, while indirect stimuli do not involve the binding of the drug to the receptor (ie, it is to the receptor via another mechanism of action). It works). The ability of a candidate drug to activate said receptor is, for example, intracellular cAMP production by overexpressing 5-HT1 BR intracellularly and before and after contacting those recombinant cells with the candidate drug. Can be evaluated by methods well known in the art, such as by measuring. Other methods for measuring the activity of 5-HT1 BR have also been described in the art, among others, reverse phase high performance liquid chromatography (HPLC-ED) coupled with an electrochemical detector using a current measurement detector. Is included. For the present invention, the agent may be selective for the 5-hydroxytryptamine 1B receptor, or to other receptors such as the 5-hydroxytryptamine 1B receptor as well as other 5-HT receptors. Can also work. In any case, as suggested herein, the promotion of self-renewal and / or differentiation of satellite cells and / or the prevention and / or inhibition of satellite cell pool depletion is 5-hydroxy by the agents of the invention. It is mediated by direct or indirect stimulation of the tryptamine 1B receptor.

According to the various aspects and embodiments of the invention described herein, "subject" or "host" means a subject having a serotoninergic system, which is more particularly particularly 5-. It contains 5-hydroxytryptamine receptors, including the hydroxytryptamine 1B receptor. Therefore, the subject preferably includes animals and humans. ,

In addition, "satellite cells", as used herein, are small, originally located under the basement membrane of muscle fibers, capable of self-renewal and the formation of new skeletal muscle cells (ie, myogenic). Means mononuclear stem cells. The most definitive marker of satellite cells is Pax7, which is present in all satellite cells of postnatal muscle and is expressed in proliferating myoblasts until they begin to differentiate. Other transcription factors common to quiescent satellite cells include Foxk1 and Pax3. Cell surface markers such as CD56 (neural cell adhesion molecule, or NCAM), hepatocyte growth factor (HGF) receptor, and c-Met can also be used to identify satellite cells from surrounding tissues. M-cadherins (Cdh15) are also generally present in quiescent satellite cells and are upregulated once they are activated. Other markers include CD106 (VCAM-1), CD34, Cindecane 3 and 4, Sox8 and Sox15. Once the satellite cells are activated, the expression of MyoD, Myf6 and Myf5 begins, followed by the expression of myogenin (MyoG), desmin, and MRF4, which undergoes differentiation into myotubes. Show that it is. The major cellular markers expressed at each stage of the muscle regeneration process are summarized in Figure 1. It is within the skill of one of ordinary skill in the art to evaluate the presence or absence of the cell marker by a method well known in the art, such as by FACS (fluorescence activated cell selection).

"Satellite cell pool" as used herein means a natural in vivo reserve or stock of satellite cells located within a subject located between the plasma membrane and basement membrane of muscle fibers. This pool consists only of quiescent (ie, dormant) satellite cells, the cell phenotype of which is Pax7 + and / or Pax3 +. In particular, the phenotypes are CD34 +, Cdh15 +, Foxk1 +, Met +, Pax3 +, Pax7 +, Sdc3 / 4 +, Sox8 +, Sox15 +, VCAM1 +, Myf5-, Myf6-, MyoD-, Desmin-, MyoG-, and MRF4-. .. More specifically, the cell phenotypes of quiescent (ie, dormant) satellite cells are CD34 +, Cdh15 +, Foxk1 +, Met +, Pax3 +, Pax7 +, Sdc3 / 4 +, Sox8 +, Sox15 +, VCAM1 +, Myf5-, Myf6-, Desmin. -, MyoG-, MRF4-, CD56 +, and MyHC-.

Thus, "depletion of the satellite cell pool" means depletion or depletion of the quiescent cells. The term "self-renewal of satellite cells" as used herein means that the cells proliferate or divide to give rise to progeny, which become resting or undergoing cell differentiation. Thus, the cell phenotypes of proliferating or self-regenerating satellite cells are Pax7 +, Myf5 +, MyoD +, Desmin-, MyoG- and MRF4-. In particular, the phenotypes are CD34 +, Cdh15 +, Foxk1 +, Met +, Pax3 +, Pax7 +, Sdc3 / 4 +, Sox8 +, Sox15 +, VCAM1 +, Myf5 +, Myf6 +, MyoD +, Desmin-, MyoG-, and MRF. More specifically, the cell phenotypes of proliferating or self-regenerating satellite cells are CD34 +, Cdh15 +, Foxk1 +, Met +, Pax3 +, Pax7 +, Sdc3 / 4 +, Sox8 +, Sox15 +, VCAM1 +, Myf5 +, Myf6 +, MyoD +, Desmin- -, MRF4-, CD56 +, and MyHC-.

The term "satellite cell differentiation" is used herein to refer to the cell from one cell phenotype to another cell phenotype for forming mature muscle fibers, more specifically from a self-regenerating cell phenotype to myoblast. It means a cellular process that changes to a cellular phenotype and then to a myoblastic phenotype. Thus, the cell phenotype of the differentiated satellite cell is desmin +, MyoG + and MRF4 +, while the cell phenotype of the fully differentiated satellite cell is MyHC +. In particular, the phenotypes of the satellite cells during differentiation are CD34 +, Cdh15 +, Foxk1 +, Met +, Pax3 +, Pax7 + Sdc3 / 4 +, Sox8 +, Sox15 +, VCAM1 +, Myf5 +, Myf6 +, MyoD +, Desmin +, MyoG +, and Desmin +, MyoG +. Can be blast cell phenotype), while fully differentiated satellite cell cell phenotypes are CD34-, Cdh15-, Foxk1-, Met-, Pax3-, Pax7-, Sdc3 / 4-, Sox8-, Sox15. -, VCAM1-, Myf5 +, Myf6 +, MyoD +, Desmin +, MyoG +, and MRF4 + and MyHC + (ie, myotube phenotype). More specifically, the cell phenotypes of the differentiating satellite cells are CD34 +, Cdh15 +, Foxk1 +, Met +, Pax3 +, Pax7 +/-, Sdc3 / 4 +, Sox8 +, Sox15 +, VCAM1 +, Myf5 +, Myf6 +, MyoD +, Desmin +, M MRF4 +, CD56 +/-, and MyHC +/- (ie, myoblast phenotype) can be, while fully differentiated satellite cell cell phenotypes are CD34-, Cdh15-, Foxk1-, Met-, Pax3. -, Pax7-, Sdc3 / 4-, Sox8-, Sox15-, VCAM1-, Myf5 +, Myf6 +, MyoD +, Desmin +, MyoG +, MRF4 +, CD56-, and MyHC + (ie, myotube phenotype).

Further definitions are given herein.

The invention can be more easily understood by reference to the following detailed description, including preferred embodiments of the invention, and examples contained herein.

We have fluoxetin or vorthioxetine, which are well-known serotonin reuptake inhibitors that increase serotonin levels and stimulate 5-HT1 BR (fluoxetin acts indirectly on 5-HT1 BR, vorthioxetine acts on 5-HT1). Upon contact with BR indirectly and directly as a partial agonist of the receptor), the satellite cells present in the damaged muscle are more active and faster than untreated animals. It was found that the division leads to the differentiation of satellite cells into mature myotubes and the emergence of new reserve quiescent satellite cells. We further improve muscle phenotype by fluoxetine in the Duchenne muscle mouse model (Mdx), in particular by reducing muscle fiber necrosis, increasing muscle fiber size, and lowering inflammatory markers. Demonstrated what was done.

Accordingly, the present invention presents novel agents for stimulating 5-HT1 BR activity to favor the proliferation and differentiation of satellite cells and prevent their in vivo depletion, thereby promoting muscle regeneration. Suggested to be used as.

Therefore, in the first aspect, the present invention is:
i) Promotes self-renewal and / or differentiation of satellite cells; and / or ii) 5-Hydroxytryptamine 1B receptor (5-hydroxytryptamine 1B receptor) for use as an agent to prevent and / or inhibit the depletion of satellite cell pools. HT1 BR) Stimulants are targeted.

As mentioned above, the agent may act directly or indirectly on the 5-HT1 BR.

More precisely, the invention is described herein for i) promoting self-renewal and / or differentiation of satellite cells and / or ii) preventing and / or inhibiting depletion of satellite cell pools. Regarding the use of 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulants.

The use may be in vivo use or in vitro use, preferably in vivo use.

More specifically, the invention relates to the manufacture of agents for i) promoting self-renewal and / or differentiation of satellite cells and / or ii) preventing and / or inhibiting depletion of satellite cell pools. Concerning the use of the 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulants described herein.

In other words, the invention is a method of i) promoting self-renewal and / or differentiation of satellite cells and / or ii) preventing and / or inhibiting depletion of the satellite cell pool, wherein an effective amount of 5 above. -A method comprising the step of administering a hydroxytryptamine 1B receptor (5-HT1 BR) stimulant to a subject in need thereof.

The invention also relates to i) the in vitro use of the 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulants described herein to promote self-renewal and / or differentiation of satellite cells.

In other words, the invention is i) an in vitro method that promotes self-renewal and / or differentiation of satellite cells in an effective amount of 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulation as described herein. The present invention relates to a method comprising the step of administering an agent to a satellite cell, particularly an isolated biological sample comprising a satellite cell pool. The biological sample can be a muscle sample herein.

"Effective amount" as used herein is the intended effect of the agents of the invention, i.e., promoting self-renewal and / or differentiation of satellite cells, and / or preventing and / or inhibiting depletion of satellite cell pools. Means to be administered in a sufficient amount to provide.

Self-renewal and / or differentiation of satellite cells, as well as satellite pool replenishment, can be assessed by analyzing the satellite cell phenotype as described above.

According to a more preferred embodiment, 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulants are affected in the present invention by loss and / or damage and / or damage of natural or pathological skeletal muscle tissue. Used in the subject who received it.

The term "spontaneous loss and / or damage and / or disorder of skeletal muscle tissue" herein includes the natural process of muscle aging resulting in progressive skeletal muscle loss. The term includes, but is not limited to, sarcopenia and related complications such as decreased muscle strength (ie, muscle weakness), fractures, and decreased motility and physical function. Generally, in humans, muscle mass begins to decrease gradually at the age of 30, but the resulting loss of strength increases exponentially with age. It is known that the prevalence of muscle loss increases from 15 to 25% under the age of 70 to over 50% in the age group over 80. Thus, in a preferred embodiment, the subject affected by natural skeletal muscle tissue loss and / or damage and / or disability is an adult subject, preferably at least 30 years old, more preferably at least 70 years old, and even more preferably. At least 80 years old. With respect to sarcopenia, Class I sarcopenia is described in the literature by Messier et al. (2009) as having an appendicular lean body mass index (ALBMI) <or = 6.44 kg. It is defined as m ^-2 (lean body mass / height of limbs), which can be identified by scanning the legs and / or arms for muscle volume. Thus, in a preferred embodiment, the subject affected by natural skeletal muscle tissue loss and / or damage and / or damage is about 6 kg / m ² or more, preferably about 6.3 kg / m ² or more, more. It preferably has an ALBMI of about 6.4 kg / m ² or more, and even more preferably about 6.44 kg / m ² or more. Sarcopenia can also be identified by performing anthropometric measurements such as brachial and crus perimeter to determine below normal levels of limb skeletal muscle (Bauer et al., 2008). Baumgartner et al. (1998) further identified sarcopenia when the skeletal muscle mass of older subjects was more than twice the standard deviation below the average for healthy young adults.

"Pathogenic skeletal muscle tissue loss and / or damage and / or disorder" as used herein is any congenital or acquired muscular degeneration, weakness, dysfunction and / or that affects skeletal muscle. It also means loss. Typical congenital illnesses that affect skeletal muscles are, but are not limited to, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, congenital muscular dystrophy, and muscular dystrophy (Limb Girdle). Includes muscular dystrophy (LGMD), facial dystrophy, muscular dystrophy, muscular dystrophy, muscular dystrophy, distal muscular dystrophy, and emery muscular dystrophy. In contrast, congenital pathologies that affect skeletal muscle are not inherited, inflammation (inflammatory muscle disorder), drugs (drug-induced muscle disorder), trauma or injury (traumatic muscle disorder, or surgery-induced muscle disorder). Can be induced by connective tissue and muscle ischemia (Burger's disease), cancer (cancer-induced sarcopenia) or even diet (dietary muscular disorder).

According to a preferred embodiment, the 5-HT1 BR stimulants used in the present invention are antidepressants and anti-migraine agents, pharmaceutically acceptable derivatives thereof, analogs, isomers, metabolites, salts, solvents. It is selected from the group consisting of solvates, inclusions, polymorphs, and cocrystals, and combinations thereof.

A "derivative" as used herein is derived directly from a chemical compound of interest (ie, a 5-HT1 BR stimulant), which is not identical but structurally similar to the compound and has the same biological activity and activity. / Or means a compound that retains physicochemical properties.

"Analogs" or "functional analogs" are herein not derived directly from the chemical compound of interest and are therefore structurally different but have the same bioactivity and / or physicochemical properties. It means a compound such as an analog indicating the above.

The "derivatives" and "analogs" of the 5-HT1 BR stimulants according to the invention retain the 5-HT1 BR stimulating activity herein as defined above, but further below. Includes compounds that do not cross the blood-brain barrier as described in.

As used herein, the term "isomer" means a compound having the same chemical formula as the compound of interest but having a different chemical structure. The term includes structural isomers and stereoisomers. If the isomers of the invention are stereoisomers, the individual stereoisomers (mirror image isomers and diastereoisomers) and mixtures thereof are included within the scope of the invention. Some compounds according to the present invention may exist in tautomeric forms (a type of structural isomer), and these are also included in the scope of the present invention.

"Metabolite" as used herein means any compound that is an intermediate and / or a product of metabolism. Metabolites from a chemical compound are usually formed within the subject to which it is administered as part of a natural biochemical process that degrades and eliminates the compound of interest. Examples of metabolites of antidepressants according to the invention are further shown below.

The term "pharmaceutically acceptable salt" or "salt", as used herein, is physiologically acceptable when used in a manner appropriate to the invention, especially when used in mammals. Means a salt that is (ie, non-toxic). Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids according to the invention are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfate. , Tartrate acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfate, benzenesulfonic acid, gluconic acid, glutamate, bis-methylenesalicylic acid, ethanedisulfonic acid, propionic acid, p-amino -Includes benzoic acid, malic acid, mandelic acid, cinnamic acid, citraconic acid, aspartic acid, stearate, palmitic acid, itaconic acid and sulfamic acid, and theophylline acetate and 8-haloteophylline, such as 8-bromoteophylline. Other acids, such as oxalic acid, are also not pharmaceutically acceptable in their own right, but can be used in the preparation of salts useful as intermediates in obtaining the compounds and their pharmaceutically acceptable acid addition salts. Salts derived from suitable bases include alkali metal salts (eg, sodium salts), alkaline earth metal salts (eg, magnesium salts), ammonium salts and N- (C1-C4 alkyl) ₄ ⁺ salts. Is done.

The term "solvate" according to the invention refers to the compound being linked to another molecule (usually a polar solvent) via non-covalent interactions, including in particular hydrates and alcoholates, such as metanolates. It should be understood to mean any form of active agent according to the invention (ie, 5-HT1 BR stimulant). Solvation methods are well known in the art.

By "inclusion" herein is meant a chemical consisting of a lattice or cage that captures or contains a second type of subject molecule / compound, which is the stability of the subject molecule / compound and in water. Can be used to increase the solubility of. The inclusions are generally polymer-based.

The term "polymorph" means various crystalline forms of a compound of interest, in which the molecules have different sequences and / or different molecular conformations. Polymorphs include crystalline liquid or crystalline solid forms of the compound of interest. Hydrate and inclusions can be polymorphs.

By "cocrystal" is used herein to mean a crystal structure composed of at least two components, where these components can be atoms, ions or molecules. Solvates and inclusions can be co-crystals under certain conditions.

With respect to the present invention, the pharmaceutically acceptable derivatives, analogs, isomers, metabolites, salts, solvates, clathrates, polymorphs, and cocrystals defined above are active forms, i.e., they. Shows 5-HT1 BR stimulating activity. The activity can be evaluated as described above.

In addition, 5-HT1 BR stimulants such as those described above, or derivatives thereof, analogs, isomers, metabolites, salts, solvates, inclusions, polymorphs, and cocrystals are preferably pharmaceutically acceptable. It should be understood that it is or is in substantially pure form. A pharmaceutically acceptable form is, among other things, a pharmaceutically acceptable level of purity, ie, conventional additives such as diluents and carriers, and any substance that are considered toxic at normal dose levels. Means to eliminate. For the present invention, the purity is preferably greater than 98%, more preferably greater than 99%, even more preferably greater than 99.9%. In a preferred embodiment, the purity is 99.9%.

As mentioned above, the 5-HT1 BR stimulants according to the invention can be selected among anti-migraine agents such as triptans or ergotamines. Triptans are well known as tryptamines used in the treatment of migraine and cluster headaches due to their agonistic effect on 5-HT1 BR and 5-HT1 DR. Examples of triptans according to the present invention include, but are not limited to, sumatriptan, lizatriptan, solmitriptan, eletriptan, almotriptan, flovatriptan, naratriptan, abitriptan, and donitriptan. Non-limiting examples of salts of the compounds include donitriptan hydrochloride, eletriptan hydrobromide, and rizatriptan benzoate.

A particularly preferred triptan according to the present invention is selected from the group consisting of sumatriptan, rizatriptan, solmitriptan, eletryptane, almotriptan, and frovatriptan.

The 5-HT1 BR stimulants according to the invention can be selected, or among antidepressants.

"Anti-depressant" as used herein means an active agent capable of treating mood disorders such as depression (including depression) and / or mood disorders. The antidepressants according to the invention are, but are not limited to, serotonin reuptake inhibitor (SRI); tricyclic antidepressant (TCA); monoamine oxidase. inhibitor) (MAO); noradrenergic and specific serotoninergic antidepressant (NaSSA); includes atypical antidepressants or antidepressant enhancers.

Serotonin reuptake inhibitors (SRIs) are compound species that generally act by inhibiting the reuptake of serotonin neurotransmitters into presynaptic terminals, thereby increasing the extracellular levels of serotonin, and thus serotoninergic transmission. Is called. Such compounds may act selectively or non-selectively on the neurotransmitter serotonin. SRI can also, in fact, exhibit varying degrees of selectivity for other monoamine reuptake systems, in particular transporters of norepinephrine and dopamine. SRIs generally include selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs) and serotonin-norepinephrine-dopamine reuptake inhibitors. (Serotonin-norepinephrine-dopamine reuptake inhibitor) (SNDRI) is included.

Examples of selective serotonin reuptake inhibitors (SSRIs) are, but are not limited to, fluoxetine, citaloplum, escitaplum, sertraline, norsertraline, paroxetine, fluboxamine, femoxetine, indalpine, alaprocrate, sericlamine, ifoxetine, zimeldine. , Dapoxetine, and etoperidone, preferably fluoxetine, citaroplum, escitaplum, sertraline, norsertraline, paroxetine, fluboxamine, femoxetine, indalpine, alaprocrate, sericramine, ifoxetine and zimeldine.

Examples of active SSRI metabolites include, but are not limited to, desmethylcitalopram, didesmethylcitalopram, and seproxetine (ie, (S) -norfluoxetine).

Examples of serotonin and norepinephrine reuptake inhibitors (SNRIs) include, but are not limited to, duloxetine, venlafaxine, desvenlafaxine, milnacipran, levomilunacipran, and sibutramine.

Examples of serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs) (also known as triple reuptake inhibitors or TRIs) are, but are not limited to, bicifadine, brasofensin, tesofencin and fomifensin, preferably. , Includes bicifadine.

Examples of tricyclic antidepressants (TCAs) according to the invention include, but are not limited to, clomipramine, amoxapine, nortriptyline, maprotiline, trimipramine, imipramine, desipramine and protriptyline.

Examples of monoamine oxidase inhibitors (MAOs) according to the invention include, but are not limited to, iproniazid, phenelzine, tranylcypromine, moclobemide, selegiline and rasagiline.

Preferably, examples of noradrenalinergic and specific serotoninergic antidepressants (NaSSA) that act by blocking presynaptic α-2 adrenergic receptors include, among others, mirtazapine, mianserin, aptazapine, esmirtazapine, setiptiline and S32212 (also known as N- [4-methoxy-3- (4-methylpiperazin-1-yl) phenyl] -1,2-dihydro-3H-benzo [e] indol-3-carboxyamide), preferably. , Mirtazapine and mianserin.

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・ｓは１または２であり；
・各Ｒ^１’は、Ｃ_１－６－アルキルにより表される基から独立に選択されるか、または２個のＲ^１’ が同じ炭素原子と結合して３～６員スピロ結合シクロアルキルを形成してもよく；
・各Ｒ^２’は、ハロゲン、シアノ、ニトロ、Ｃ_１－６－アルク（エン／イン）イル、Ｃ_１－６－アルク（エン／イン）イルオキシ、Ｃ_１－６－アルク（エン／イン）イルスルファニル、ヒドロキシ、ヒドロキシ－Ｃ_１－６－アルク（エン／イン）イル、ハロ－Ｃ_１－６－アルク（エン／イン）イル、ハロ－Ｃ_１－６－アルク（エン／イン）イルオキシ、Ｃ_３－８－シクロアルク（エン）イル、Ｃ_３－８－シクロアルク（エン）イル－Ｃ_１－６－アルク（エン／イン）イル、アシル、Ｃ_１－６－アルク（エン／イン）イルオキシカルボニル、Ｃ_１－６－アルク（エン／イン）イルスルホニル、または－ＮＲｘＲｙ；－ＮＲｘＣＯ－Ｃ_１－６－アルク（エン／イン）イルにより表される基から独立に選択され；
・各Ｒ^３’は、ハロゲン、シアノ、ニトロ、Ｃ_１－６－アルク（エン／イン）イル、Ｃ_１－６－アルク（エン／イン）イルオキシ、Ｃ_１－６－アルク（エン／イン）イルスルファニル、ヒドロキシ、ヒドロキシ－Ｃ_１－６－アルク（エン／イン）イル、ハロ－Ｃ_１－６－アルク（エン／イン）イル、ハロ－Ｃ_１－６－アルク（エン／イン）イルオキシ、Ｃ_３－８－シクロアルク（エン）イル、Ｃ_３－８－シクロアルク（エン）イル－Ｃ_１－６－アルク（エン／イン）イル、Ｃ_１－６－アルク（エン／イン）イルスルホニル、アリール、Ｃ_１－６－アルク（エン／イン）イルオキシカルボニル、アシル、－ＮＲ^ｘＣＯ－Ｃ_１－６－アルク（エン／イン）イル、ＣＯＮＲ^ｘＲ^ｙまたはＮＲ^ｘＲ^ｙにより表される基から独立に選択され；
あるいは、２個の隣接するＲ^３’置換基は一緒に、

（式中、ＷはＯまたはＳであり、かつ、Ｒ^ａおよびＲ^ｂは水素またはＣ_１－６－アルキルである）
からなる群から選択されるフェニル環と縮合した複素環を形成するか、
あるいは、２個の隣接するＲ^３’置換基は一緒に、１、２または３個のヘテロ原子を含有する縮合複素芳香族系を形成し、
各Ｒ^ｘおよびＲ^ｙは、水素、Ｃ_１－６－アルク（エン／イン）イル、Ｃ_３－８－シクロアルク（エン）イル、Ｃ_３－８－シクロアルク（エン）イル－Ｃ_１－６－アルク（エン／イン）イル、もしくはアリールにより表されるから独立に選択され；またはＲ^ｘ およびＲ^ｙは、それらが結合している窒素と一緒に、場合により１個のさらなるヘテロ原子を含有してもよい３～７員環を形成し、
またはその薬学上許容可能な塩である。 Examples of atypical antidepressants (thus defined as not belonging to any of the above antidepressant species) or antidepressant enhancers are, but are not limited to, bisarylsulfanylamines such as vortioxetine, as well. Includes tianeptine, agomelatine, nephazodon, trazodon, buspirone, tandospirone, and ketamine, preferably vortioxetine, thianeptine, agomelatine, nephazodon, trazodon, buspirone, tandospirone, and ketamine.
Bisarylsulfanylamines are disclosed in patent application WO2003 / 029232, which is incorporated by reference, and is within the scope of 5-HT1 BR stimulants according to the invention. The compound can be described according to the following general formula (A).

During the ceremony
・ M is 1 or 2;
P is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
Q is 0, 1, 2, 3 or 4;
• S is 1 or 2;
Each R ^1'is selected independently of the group represented by C _1-6 -alkyl, or two R ¹ 's are bonded to the same carbon atom to form a 3- to 6-membered spiro-bonded cycloalkyl. May be formed;
-Each R ^2'is halogen, cyano, nitro, C _1-6 -alk (en / in) yl, C _1-6 -alk (en / in) yloxy, C _1-6 -alk (en / in). Ilsulfanyl, hydroxy, hydroxy-C _1-6 -alk (en / in) yl, halo-C _1-6 -alk (en / in) yl, halo-C _1-6 -alk (en / in) yloxy, C _3-8 -cycloalk (enyne) yl, C _3-8 -cycloalk (en) yl-C _1-6 -alk (en / in) yl, acyl, C _1-6 -alk (en / in) Selected independently from the groups represented by yloxycarbonyl, C _1-6 -alk (ene / in) ylsulfonyl, or -NRxCO-C _1-6 -alk (en / in) yl;
-Each R ^3'is halogen, cyano, nitro, C _1-6 -alk (en / in) yl, C _1-6 -alk (en / in) yloxy, C _1-6 -alk (en / in). Ilsulfanyl, hydroxy, hydroxy-C _1-6 -alk (en / in) yl, halo-C _1-6 -alk (en / in) yl, halo-C _1-6 -alk (en / in) yloxy, C _3-8 -cycloalk (en) yl, C _3-8 -cycloalk (en) yl-C _1-6 -alk (en / in) yl, C _1-6 -alk (en / in) yl sulfonyl , Aryl, C _1-6 -alk (en / in) yloxycarbonyl, acyl, -NR ^x CO-C _1-6 -alk (en / in) yl, CONR ^x R ^y or NR ^x R ^y Selected independently of the group;
Alternatively, two adjacent ^{R3'substituents} together,

(In the formula, W is O or S, and Ra and R ^b are hydrogen or C _1-6 -alkyl ⁾ .
Form a heterocycle condensed with a phenyl ring selected from the group consisting of
Alternatively, two adjacent ^{R3'substituted} groups together form a condensed complex aromatic system containing 1, 2 or 3 heteroatoms.
Each R ^x and R ^y are hydrogen, C _1-6 -alk (en / in) yl, C _3-8 -cyclo alk (en) yl, C _3-8 -cyclo alk (en) yl-C _{1- 6} -Selected independently from represented by alc (ene / in) yl, or aryl; or ^Rx and ^Ry , together with the nitrogen to which they are attached, optionally one additional heteroatom. Forming a 3- to 7-membered ring that may be contained,
Or its pharmaceutically acceptable salt.

The synthesis of the compound of the general formula (A) is well described in WO2003 / 029232 and does not need to be described in detail herein.

In a preferred embodiment of the general formula (A), p is 0.

In a preferred embodiment of the general formula (A), m is 1 or 2.

In a preferred embodiment of the general formula (A), R ^2'is trifluoromethyl, or C _1-6 -alkyl.

In a preferred embodiment of the general formula (A), R ^3'is selected from the group consisting of halogen, C _1-6 -alkoxy, C _1-6 -sulfanyl, C _1-6 -alkylhydroxy and trifluoromethyl. ..

In a more preferred embodiment of the general formula (A), m = 1, p = 0, q = 0, R ^3'is methyl and s = 2.

１－［２－（３，５－ジメチルフェニルスルファニル）フェニル］ピペラジン、
１－［２－（２，６－ジメチルフェニルスルファニル）フェニル］ピペラジン、
１－［２－（２，５－ジメチルフェニルスルファニル）フェニル］ピペラジン、
１－［２－（２－トリフルオロメチルフェニルスルファニル）フェニル］［１，４］ジアゼパン、
１－［２－（３－メチルフェニルスルファニル）フェニル］－［１，４］－ジアゼパン、
２－（４－メチルフェニルスルファニル）フェニル－１－ピペラジン、
１－［２－（４－クロロフェニルスルファニル）フェニル］－ピペラジン、
１－［２－（４－メトキシフェニルスルファニル）－４－クロロフェニル］ピペラジン、
１－［２－（４－メトキシフェニルスルファニル）－４－メチルフェニル］ピペラジン、
１－［２－（４－メトキシフェニルスルファニル）－５－メチルフェニル］ピペラジン、
１－［２－（４－フルオロフェニルスルファニル）－５－メチルフェニル］ピペラジン、
１－［２－（４－メトキシフェニルスルファニル）－５－トリフルオロメチルフェニル］ピペラジン、
１－［２－（４－クロロフェニルスルファニル）フェニル］－３－メチルピペラジン、
１－［２－（４－クロロフェニルスルファニル）フェニル］－３，５－ジメチルピペラジン、
またはそれらの薬学上許容可能な塩。 In a particularly preferred embodiment of the general formula (A), the compound of the formula (A) is any of the following:
1- [2- (2-trifluoromethylphenylsulfanyl) phenyl] piperazine,
1- [2- (4-bromophenylsulfanyl) phenyl] piperazine,
1- {2- [4- (methylsulfanyl) phenylsulfanyl] phenyl} piperazine,
1- [2- (4-Hydroxyphenylsulfanyl] phenyl} piperazine,
1- [2- (2,4-dimethylphenylsulfanyl) phenyl] piperazine (also known as vortioxetine),
Vortioxetine:

1- [2- (3,5-dimethylphenylsulfanyl) phenyl] piperazine,
1- [2- (2,6-dimethylphenylsulfanyl) phenyl] piperazine,
1- [2- (2,5-dimethylphenylsulfanyl) phenyl] piperazine,
1- [2- (2-trifluoromethylphenylsulfanyl) phenyl] [1,4] diazepan,
1- [2- (3-Methylphenylsulfanyl) phenyl]-[1,4] -diazepan,
2- (4-Methylphenylsulfanyl) Phenyl-1-piperazine,
1- [2- (4-chlorophenylsulfanyl) phenyl] -piperazine,
1- [2- (4-Methoxyphenylsulfanyl) -4-chlorophenyl] piperazine,
1- [2- (4-Methoxyphenylsulfanyl) -4-methylphenyl] piperazine,
1- [2- (4-Methoxyphenylsulfanyl) -5-methylphenyl] piperazine,
1- [2- (4-Fluorophenylsulfanyl) -5-methylphenyl] piperazine,
1- [2- (4-Methoxyphenylsulfanyl) -5-trifluoromethylphenyl] piperazine,
1- [2- (4-chlorophenylsulfanyl) phenyl] -3-methylpiperazine,
1- [2- (4-chlorophenylsulfanyl) phenyl] -3,5-dimethylpiperazine,
Or their pharmaceutically acceptable salts.

In the most preferred embodiment, the compound of formula (A) is 1- [2- (2,4-dimethylphenylsulfanyl) phenyl] piperazine (ie, vortioxetine).

"Halogen" as used herein means fluoro (F), chloro (Cl), bromo (Br) or iodine (I).

"Alkyl", "alkenyl", "alkynyl", and "aryl" are further defined below.

The expression C _1-6 -alk (en / in) yl means a C _1-6 -alkyl, C _2-6 -alkenyl or C _2-6 -alkynyl group.

The expression C _3-8 -cycloalk (en) yl means C _3-8 -cycloalkyl- or cycloalkenyl group.

The term C _1-6 alkyl means branched or unbranched alkyl with 1-6 carbon atoms and is, but not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl. , 2-Butyl, 2-Methyl-2-propyl and 2-Methyl-1-propyl.

Similarly, C _2-6 alkenyl and C _2-6 alkynyl represent and limit such groups each having 2 to 6 carbon atoms containing 1 double bond and 1 triple bond, respectively. Although not included, it includes ethenyl, propenyl, butenyl, ethynyl, propynyl and butynyl.

The term C _3-8 cycloalkyl represents a monocyclic or bicyclic carbocycle having 3-8 C atoms and includes, but is not limited to, cyclopropyl, cyclopentyl, cyclohexyl and the like.

The term C _3-8 cycloalkenyl represents a monocyclic or bicyclic carbocycle having 3-8 C atoms and containing one double bond.

In the term C _3-8 -cycloalk (en) yl-C _1-6 -alk (en / in) il, C _3-8 -cycloalk (en) il and C _1-6 -alk (en / in) il. ) Il is as defined above.

C _1-6 -alk (en / in) yloxy, C _1-6 alk (en / in) ylsulfanyl, hydroxy-C _1-6 -alk (en / in) yl, halo-C _1-6 -alk ( Terms such as en / in) yl, halo-C _1-6 -alk (en / in) yloxy, C _1-6 -alk (en / in) ylsulfonyl are referred to as C _1-6 -alk (en / in). Il represents such a group, as defined above.

As used herein, the term C _1-6 -alk (en / in) yloxycarbonyl means the group of formula C _1-6 -alk (en / in) yl-O-CO-. Here, C _1-6 -arc (en / in) ill is as defined above.

As used herein, the term acyl refers to formyl, C _1-6 -alk (en / in) ylcarbonyl, arylcarbonyl, aryl-C _1-6 -alk (en / in) ylcarbonyl, C _{3 -8} -cycloalk (en) ylcarbonyl or C _3-8 -cycloalk (en) yl-C _1-6 -alk (en / in) means yl-carbonyl group.

The term 3- to 7-membered ring, which may optionally contain one additional heteroatom, as used herein, 1-morpholinyl, 1-piperidinyl, 1-azepinyl, 1-piperazinyl, 1-homopiperazinyl. , 1-imidazolyl, 1-pyrrolyl or pyrazolyl and the like, all of which may be further substituted with C _1-6 -alkyl.

The heterocycle formed by two adjacent ^{R3'substituted} groups and fused with the parent ring together is 3H-1,2,3-oxathiazole, 1,3,2-oxathiazole, 1,3,2. -Dioxazole, 3H-1,2,3-dithiazole, 1,3,2-dithiazole, 1,2,3-oxadiazole, 1,2,3-thiadiazol, 1H-1,2,3-triazole, 5-membered monocyclic rings such as isoxazole, oxazole, isothiazole, thiazole, 1H-imidazole, 1H-pyrazole, 1H-pyrrole, furan or thiophene and 1,2,3-oxathiazine, 1,2,4-oxathiazine, 1 , 2,5-oxathiazine, 1,4,2-oxathiazine, 1,4,3-oxathiazine, 1,2,3-dioxazine, 1,2,4-dioxazine, 4H-1,3,2-dioxazine, 1 , 4,2-dioxazine, 2H-1,5,2-dioxazine, 1,2,3-dithiadin, 1,2,4-dithiadin, 4H-1,3,2-dithiadin, 1,4,2-dithiadin , 2H-1,5,2-dithiadin, 2H-1,2,3-oxadiadin, 2H-1,2,4-oxadiadin, 2H-1,2,5-oxadiadin, 2H-1,2,b-oxadiadin , 2H-1,3,4-oxadiadin, 2H-1,2,3-thiadiadin, 2H-1,2,4-thiadiadin, 2H-1,2,5-thiadiadin, 2H-1,2,6-thiadiadin , 2H-1,3,4-thiadiadin, 1,2,3-triazine, 1,2,4-triazine, 2H-1,2-oxadin, 2H-1,3-oxadin, 2H-1,4-oxadin , 2H-1,2-thiadin, 2H-1,3-thiadin, 2H-1,4-thiadin, pyrazine, pyridazine, pyrimidine, 4H-1,3-oxatidine, 1,4-oxatidine, 4H-1,3 -A ring such as a 6-membered monocyclic ring such as dioxyin, 1,4-dioxyin, 4H-1,3-dithione, 1,4-dithione, pyridine, 2H-pyran or 2H-thiin may be formed.

Further, the compound of the general formula (A) may exist in a non-solvate form and in a solvate form with a pharmaceutically acceptable solvent such as water or ethanol.

Some compounds of the general formula (A) contain a chiral center, and such compounds exist in the form of isomers (ie, enantiomers). Any mixture thereof, including such isomers and racemic mixtures, is within the scope of the invention.

Particularly preferred antidepressants according to the invention are bisarylsulfanylamines such as those described above, such as vortioxetine and fluoxetine, citaloplum, escitaplum, sertraline, paroxetine, fluboxamine, femoxetine, indalpine, arapolate, dimeridine, duroxetine, venlafaxine. Desvenlafaxin, Milnashiplan, Levomilnaciplan, Cibutramine, Bisifadine, Chromipramine, Amoxapine, Maprotyrin, Imipramine, Decipramine, Moclobemide, Selegiline, Agomelatine, Mianserin, Thianeptine, Agomelatine, Trazodon, Buspilon, Selected from the group. More preferably, the antidepressant according to the invention is selected from the group consisting of bisarylsulfanylamines as described above, such as vortioxetine, and fluoxetine.

Other suitable 5-HT1 BR stimulants according to the present invention include ampyltoline hydrochloride, CGS-12066A, CGS 12066B dimaleate, oxymethazoline, 5-carboxamidetryptamine, CP-93129 and CP-93129 dihydrochloride. The salt, its salt such as CP-94253 and CP-94253 hydrochloride, its salt such as CP-122,288, CP-135,807, RU-24969 and RU-24969 hemosuccinate, ziplacidone, acenapin, 5- It can be nonyloxytryptamine oxalate, pindrol and (S)-(-)-pindrol.

According to a preferred embodiment, the 5-HT1 BR stimulant used in the present invention is an atypical antidepressant and an antidepressant selected from the group consisting of SRIs, in particular SSRIs.

More preferably, the 5-HT1 BR stimulant of the present invention is the atypical antidepressant vortioxetine or SSRI fluoxetine. Most preferably, the 5-HT1 BR stimulant of the present invention is vortioxetine.

However, if the 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant of the present invention exhibits undesired CNS-related adverse effects, it is particularly advantageous to limit the action of the agent on the peripheral serotonin system. be. Antidepressants are particularly well known to exhibit these side effects. Side effects can be prevented by chemically modifying the structure of the agent and, in particular, by grafting a charged chemical moiety so that it does not cross the blood-brain barrier.

Thus, in a preferred embodiment, the 5-HT1 BR stimulant used in the present invention is preferably modified to include at least one positively charged, charged chemical moiety. In particular, this positive charge can be retained in a wide range of pH, especially physiological pH.

In other words, such modified 5-HT1 BR stimulants according to the invention cannot cross the blood-brain barrier. Thus, the antidepressants and anti-migraine agents modified in this way do not have antidepressant and anti-migraine abilities, respectively.

Such chemical modifications are described in detail in patent application WO 2007/148341, which is incorporated herein by reference, and 5-HT1 of those compounds while preventing them from crossing the blood-brain barrier. It can be performed to retain BR stimulating activity.

The terms "charged chemical moiety," "charged moiety," "charged chemical group," or "charged group," as used herein, form part of an organic molecule that produces positive or negative electrostatic charges. It means a characteristic atom or group of atoms.

Thus, the "positively charged chemical moiety", "positively charged moiety", "positively charged chemical group" or "positively charged group" means the charged chemical moiety as defined above, characterized by a positive electrostatic charge. do. A compound containing one or more positively charged moieties is a molecular ion, often referred to as a molecular cation. A positively charged atomic group has at least one electron less than the number of protons in these atoms. Positively charged chemical moieties include, but are not limited to, ammonium and sulfonium groups.

A positively charged group that retains its charge at physiological pH is a group that cannot participate in electron exchange interactions in the pH range typical of the physiological environment in the body where the 5-HT1-BR stimulant is active. Generally, the physiological pH is about 7.4, so a positively charged group that retains its charge at physiological pH means a positively charged chemical group that maintains ionization in the pH range of about 5-8. The modified 5-HT1-BR stimulants according to the invention are adversely affected by the pH level of the GI, as the positively charged chemical moieties according to the invention remain positively charged even in GIs where the pH level is extremely low with respect to physiological pH. Please note that you will not receive.

Furthermore, according to a further preferred embodiment, the positively charged chemical moiety is a quaternary ammonium group or a tertiary sulfonium group.

As used herein, "quaternary ammonium" means a nitrogen atom that is attached to four non-hydrogen substituents and thus forms part of a positively charged molecule (amine). The term "amine" refers to a -NR'R "group in which each of R'and R" is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic, aryl or heteroaryl.

The "tertiary sulfonium group" means a sulfur atom that is attached to three non-hydrogen substituents and thus forms part of a positively charged molecule (sulfonium). The term "sulfonium" means −S ⁺ R'R'' in which R'and R'' are independently alkyl, cycloalkyl, heteroalicyclic, aryl or heteroaryl, respectively.

According to the present invention, the term "alkyl group" means a linear or branched saturated aliphatic group. Preferably, the alkyl group has 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. For example, when a numerical range such as "1 to 10" is shown herein, it is 10 from 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. whose group (in this case, an alkyl group) is 1 carbon atom. It means that it can contain less than one carbon atom. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, tert-butyl and isopropyl groups. Alkyl groups can be further substituted. When substituted, the substituents can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, halide, hydroxy, alkoxy and hydroxyalkyl. The term "alkyl", as used herein, further includes saturated or unsaturated hydrocarbons, and thus the term further includes alkenyl and alkynyl.

The term "cycloalkyl" means an aliphatic monocyclic or bicyclic ring having 3 to 8 carbon atoms, including, but not limited to, cyclopropyl, cyclopentyl, cyclohexyl and the like.

The term "alkenyl" is unsaturated as defined above, which can be substituted with at least two carbon atoms and at least one carbon-carbon double bond and with one or more substituents as described above. Includes alkyl. The term "alkynyl" is an unsaturated alkyl that has at least two carbon atoms and at least one carbon-carbon triple bond and can be substituted with one or more substituents as described above.

The term "aryl" refers to a fused ring polycyclic (ie, a ring sharing adjacent carbon atom pairs) groups having a full carbon monocyclic or fully conjugated pi-electron system.

As used herein, an "aryl or heteroaryl group" is a monocyclic or polycyclic aromatic comprising preferably between 4 and 15 carbon atoms, preferably between 5 and 10 carbon atoms. It means a family group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and the like. The aryl group according to the present invention may be further substituted with one or more substituents as described above. A heteroaryl group generally comprises at least one heteroatom such as nitrogen, oxygen, and sulfur, where the heteroatom is any atom that is not carbon or hydrogen. Examples of heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be further substituted with one or more substituents as described above, with typical examples being thiadiazole, pyridine, pyrrole, oxazole, indole, and purine.

In a more preferred embodiment, the quaternary ammonium group is of formula (I).
-(NR ₁ R ₂ R ₃ ) ^{+ Z-} (I)
In the formula,
Z is an organic or inorganic anion such as NO _3- ^, H ₂ PO ₄ ^2- , Br-, HSO _4- ^, CH ₃ SO _3- ^or tartrate anion; and R ₁ , R ₂ and R ₃ Are independently selected from the group consisting of alkyl, aryl and cycloalkyl, respectively.

Preferably, R ₁ , R ₂ and R ₃ are alkyls each having 1 to 4 carbon atoms, and more preferably R ₁ , R ₂ and R ₃ are methyl and each have a positively charged group. It either arises or is a quaternary ammonium group- (NMe ₃ ) ⁺ .

In another preferred embodiment, the tertiary sulfonium group is of formula (II).
-(SR ₄ R ₅ ) ^{+ Z-} (II)
In the formula,
Z is an organic or inorganic anion such as NO _3- ^, H ₂ PO ₄ ^2- , Br-, HSO _4- ^, CH ₃ SO _3- ^or tartrate anion; and R ₄ and R ₅ are alkyl, respectively. , Independently selected from the group consisting of aryl and cycloalkyl.

Preferably, R ₄ and R ₅ are alkyls each having 1 to 4 carbon atoms, more preferably R ₄ and R ₅ are each methyl and give rise to a positively charged group, or sulfonium- (. SMe ₂ ) ⁺ .

Positively charged groups are from existing groups that form part of the 5-HT1 BR stimulant on the 5-HT1 BR stimulant, i.e., converting partially charged or uncharged groups to positively charged groups. Alternatively, the existing positively charged groups that can participate in the proton exchange interaction are changed to those that cannot participate in such an interaction, making it an irreversible positive charge or a permanent positive charge, thereby modifying the 5-HT1 BR stimulant. It can be formed by doing.

Alternatively, a positively charged group can be obtained by substituting one or more carbon atoms with a positively charged group, eg, by substituting a hydrogen atom or any other substituent with a quaternary ammonium group or a tertiary sulfonium group. It can also be added to a 5-HT1 BR stimulant.

Examples of preferred 5-HT1 BR stimulants from which the compounds described herein can be derived are, but are not limited to, bisarylsulfanylamines such as those described above, such as vortioxetine, as well as fluoroxetine, citaloplum, alaprocrat. , Dapoxetine, fluboxamine, paroxetine, sertraline, benrafaxin, zimeldine, etoperidone, densvalafaxine, duroxetine, milnaciplan, nephazodone, benrafaxin, brasofensin, tesofencin and violet, preferably vortioxetine. Includes fluoxetine, citaloplum, alaprocrate, dapoxetine, fluboxamine, paroxetine, sertraline, benrafaxin and zimeldine. In fact, all of these agents already contain at least one amine group, which can be easily converted to a quaternary ammonium, i.e., a positively charged group as defined above. In particular, the agent can be modified to contain at least one quaternary ammonium group of formula (I) as described above.

An example of a derivative of citalopram comprising such a quaternary ammonium group is n-methyl-citalopram (NMC), the synthesis of which is described in detail in patent application WO 2007/128341.

Bisarylsulfanylamines of formula (A) also contain sulfur groups as well as amine groups herein, in particular, and are readily converted to quaternary ammonium groups and / or tertiary sulfonium groups, respectively. It is advantageous because it can be done. The positively charged moiety can also be attached to the carbon atom of the piperazine group of the compound.

式中、
・Ｚは、上記に定義される有機もしくは無機陰イオン、例えば、ＮＯ_３ ^－、Ｈ_２ＰＯ_４ ^２－、Ｂｒ－、ＨＳＯ_４ ^－、ＣＨ_３ＳＯ_３ ^－、もしくは酒石酸陰イオン、または有機もしくは無機陰イオンの混合物であり、その全体の電荷は式（Ｂ）の化合物が中性となるものであり；
・Ｒ^１’、Ｒ^２’、Ｒ^３’、ｍ、ｐ、ｑおよびは上記に定義される通りであり、ここで、Ｒ^３’は場合により、上記に定義されるアンモニウム基またはスルホニウム基で置換されたＣ_１－６－アルク（エン／イン）イルオキシ基であってよく、好ましくは、Ｒ^３’はコリンであり；
・ｔは０、１、２、３、４、５、６、７または８であり、好ましくは、０または１、より好ましくは、０であり、ただし、ｔ＋ｐ≦８であり；
・各Ｒ^４’は、同一または異なる、好ましくは、上記に定義されるように正に荷電した少なくとも１つの荷電化学部分、例えば、スルホニウム基またはアンモニウム基であり；
・Ｘ’は、
○ －（ＮＲ^５’Ｒ^６’）^＋－、ここで、
Ｒ^５’およびＲ^６’はそれぞれ独立に、本明細書に定義される水素、アルキル、アリールおよびシクロアルキルにより、好ましくは、水素、Ｃ_１－６－アルキル、およびＣ_３－８－シクロアルキルにより表される基から選択されるか；あるいは
Ｒ^５’Ｒ^６’はそれらが結合している窒素と一緒にシクロヘテロアルキル、好ましくは、３～８員シクロヘテロアルキル、より好ましくは、３～６員シクロヘテロアルキルを形成する；
○ －ＮＨ－；
○ －ＮＲ^７’－、ここで、Ｒ^７’はＣ_１－６－アルキルであり；
○ －Ｎ^＋（Ｏ^－）Ｒ^８’－、ここで、Ｒ^８’はＣ_１－６－アルキルであり；
○ －ＮＣ（Ｏ）Ｒ^９’－、ここで、Ｒ^９’はアミノ酸（前記アミノ酸は、ヒスチジン、アルギニンもしくはリシンなど、好ましくは正に荷電している）、またはアミノ酸誘導体（前記誘導体は、コリンもしくはカルニチンなど、好ましくは正に荷電している）、またはＣ_１－６－アルキルホスホニウムである；
からなる群から選択され；
・Ｙ’は、
○ －Ｓ－；
○ －（ＳＲ^１０’）^＋－、ここで、Ｒ^１０’は、本明細書に定義される水素、アルキル、アリールおよびシクロアルキルにより表される基から選択され、好ましくは、Ｃ_１－６－アルキルである；および
○ －Ｓ^＋（Ｏ）^－－
からなる群から選択される。 A particularly preferable derivative of the bisarylsulfanylamine is a compound of the following formula (B).

During the ceremony
Z is an organic or inorganic anion as defined above, eg, NO _3- ^, H ₂ PO ₄ ^2- , Br-, HSO _4- ^, CH ₃ SO _3- ^, or tartrate anion, or organic or inorganic. It is a mixture of anions, the total charge of which is that the compound of formula (B) is neutral;
R ^1' , R ^2' , R ^{3' ,} m, p, q and are as defined above, where R 3'is optionally an ammonium or sulfonium group as defined above. It may be a substituted C _1-6 -alk (en / in) yloxy group, preferably R ^3'is choline;
T is 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably 0 or 1, more preferably 0, but t + p ≦ 8;
Each R ^4'is the same or different, preferably at least one charged chemical moiety positively charged as defined above, eg, a sulfonium group or an ammonium group;
・ X'is
○-(NR 5'R ^{6' ) +} -, here,
R ^5'and R ^6'are independently by hydrogen, alkyl, aryl and cycloalkyl as defined herein, preferably by hydrogen, C _1-6 -alkyl, and C _3-8 -cycloalkyl, respectively. Is it selected from the groups represented; or R ^{5'R 6'is} cycloheteroalkyl, preferably 3-8 membered cycloheteroalkyl, more preferably 3-6, together with the nitrogen to which they are attached. Forming member cycloheteroalkyl;
○ -NH-;
○ -NR ^7' -where R ^7'is C _1-6 -alkyl;
○ -N ⁺ ( ^O- ) R ^8'- , where R ^8'is C _1-6 -alkyl;
-NC (O) R ^9' -where R ^9'is an amino acid (the amino acid is preferably positively charged, such as histidine, arginine or lysine) or an amino acid derivative (the derivative is choline). Alternatively, it is preferably positively charged, such as carnitine), or C _1-6 -alkylphosphonium;
Selected from the group consisting of;
・ Y'is
○ -S-;
-(SR ^{10' ) +} -, where R 10'is selected from the groups represented herein by hydrogen, alkyl, aryl and cycloalkyl, preferably C _1-6- . Alkyl; and ○ －S ⁺ (O) ^－ －
It is selected from the group consisting of.

One of skill in the art will readily appreciate that anion Z exists to balance the positive charge of the molecule. Thus, the compound of formula (B) comprises the number of anions Z required to cancel the positive charge of the molecule. Those skilled in the art will further understand that if p> 0 and t> 0, then R ^1'and R ^4'are attached to any of the different carbon but carbon atoms of the heterocyclic ring. There will be.

In a preferred embodiment, only one of X', Y', R ^4' (if t> 0) and R ^3' (if substituted with an ammonium or sulfonium group) is the positively charged chemical moiety. ..

Preferred embodiments for R ^1' , R ^2' , R ^3' , m, p, q and s are defined above.

In a preferred embodiment of the invention, the 5-HT1 BR stimulant in which the compounds described herein are induced such that they particularly preferably contain a charged chemical moiety is such as bisarylsulfanylamine as defined above. It is selected from the group consisting of atypical antidepressants and SRIs, in particular SSRIs.

More preferably, the modified 5-HT1 BR stimulant is the atypical antidepressant vortioxetine or SSRI fluoxetine. Most preferably, the modified 5-HT1 BR stimulant is vortioxetine.

式中、Ｚ、ｔ、Ｒ^４’、Ｘ’およびＹ’は上記に定義される通りである。より好ましくは、ｔ＝０である。 For example, vortioxetine can be chemically modified as follows:

In the formula, Z, t, R ^4' , X'and Y'are as defined above. More preferably, t = 0.

［式中、ＨＺは好ましくはＨＮＯ_３、Ｈ_３ＰＯ_４、ＨＢｒ、Ｈ_２ＳＯ_４、ＣＨ_３ＳＯ_３Ｈ、または酒石酸（ボルチオキセチンの塩）である]；

（式中、ＡＡはアミノ酸である）、
好ましくは、

（式中、ＡＡ＋は、ヒスチジン、アルギニンまたはリシンなどの正荷電アミノ酸である）；

からなる群から選択される。 Particularly preferred salts, derivatives and / or analogs of vortioxetine, preferably comprising at least one positively charged charged chemical moiety, are

[In the formula, HZ is preferably HNO ₃ , H ₃ PO ₄ , HBr, H ₂ SO ₄ , CH ₃ SO ₃ H, or tartaric acid (a salt of vortioxetine)];

(AA is an amino acid in the formula),
Preferably,

(In the formula, AA + is a positively charged amino acid such as histidine, arginine or lysine);

It is selected from the group consisting of.

Preferably, said salt, derivative and / or analog of vortioxetine comprises at least one positively charged charged chemical moiety.
-Vortioxetine salt as described above;
Vortioxetine coupled to a positively charged amino acid (preferably at least one) such as histidine, arginine or lysine as described above;
・ Pyrrolidinium-vortioxetine;
・ Piperadinium-vortioxetine;
-Dimethylammonium-vortioxetine;
・ Sulfonium-vortioxetine;
N-oxide-vortioxetine;
-Sulfoxide-vortioxetine;
-Phoenium-vortioxetine; and-Tempol-carbamate-vortioxetine.

More preferably, said salts, derivatives and / or analogs of vortioxetine comprising at least one positively charged chemical moiety.
-Vortioxetine salt as described above;
Vortioxetine coupled to a positively charged amino acid (preferably at least one) such as histidine, arginine or lysine as described above;
・ Pyrrolidinium-vortioxetine;
・ Piperadinium-vortioxetine;
-Dimethylammonium-vortioxetine;
・ Sulfonium-vortioxetine;
N-oxide-vortioxetine;
• Sulfoxide-vortioxetine; and • Phosnium-vortioxetine.

Even more preferably, said salts, derivatives and / or analogs of vortioxetine comprising at least one positively charged chemical moiety.
-Vortioxetine salt as described above;
Vortioxetine coupled to a positively charged amino acid (preferably at least one) such as histidine, arginine or lysine as described above;
・ Pyrrolidinium-vortioxetine;
・ Piperadinium-vortioxetine;
-Dimethylammonium-vortioxetine;
-Selected from the group consisting of sulfonium-vortioxetine; and-phosphonium-vortioxetine.

Even more preferably, the positively charged vortioxetine is selected from the group consisting of histidine-vortioxetine and pyrrolidinium-vortioxetine.

The above compounds can be produced according to conventional methods in the art. Such a method is described in more detail below.

For example, one of ordinary skill in the art can take the following steps to synthesize pyrrolidinium-vortioxetine or piperazinium-vortioxetine.

More specifically, for the formation of pyrrolidinium (n = 1) on 4-arylpiperazine, the reaction is K2 CO _{3 ,} ethanol (EtOH) and reflux for 10 hours (part of the specification by reference). _Mokrosz et al., 1992), or using either K2 CO ₃ , acetone and 15 hours of reflux (see WO2004 / 9914A1, which is incorporated herein by reference). be able to.

Further, for example, to synthesize dimethylammonium-vortioxetine, those skilled in the art can take the following procedure.

More specifically, for 4-arylpiperazine dimethylation, Romanelli et al. (2001) (incorporated herein by reference) can be referred to, the first step of which is Eschweiler-Clark. (Eschweiler-clarke) Reaction, the second step is methylation.

As another embodiment, those skilled in the art can take the following steps to synthesize amino acid derivatives of vortioxetine, choline-vortioxetine and carnitine-vortioxetine.

More specifically, for amide formation on carnitine, the reaction is here on acylhydrazine with pyridine, ethylenedichloride (EDC), dimethylformamide (DMF), and ethanol (EtOH). Kuroda et al., 1996) as part, or pyridine, ethylene dichloride (EDC), methanol (Methanol) and acetonitrile (CH ₃ CN) on primary amines (cited herein by reference). It can be done by Nakaya et al., 2001), which is a part of.

As another embodiment, those skilled in the art can take the following steps to synthesize phosphonium-vortioxetine.

As another embodiment, those skilled in the art can take the following steps to synthesize N-oxide-vortioxetine.

More specifically, for N-oxidation of 4-aryl-piperazine, the reaction can be carried out at 20-45 ° C. with meta-chloroperoxybenzoic acid (m-CPBA) and CH ₂ Cl ₂ (cited). US 2008/153812A1, WO2011 / 162515A2 or WO2004 / 104007A1 which are incorporated herein by reference).

As another embodiment, those skilled in the art can take the following steps to synthesize tempo-carbamate-vortioxetine.

As another embodiment, those skilled in the art can take the following steps to synthesize benzyl-choline-vortioxetine.

More specifically, for benzylic bromination, the reaction is N-bromosuccinimide, azobisisobutyronitrile (AIBN) and tetrachloromethane (CCl ₄ ) (US2010, which is incorporated herein by reference). / 4159A1), or N-bromosuccinimide, meta-chloroperoxybenzoic acid (m-CPBA) and tetrachloromethane (CCl ₄ ) (Farmaco, 1989, 44, incorporated herein by reference). It can be done by using any of (see from p.683).

The 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant as defined above is administered simultaneously, individually or sequentially to a pharmaceutical composition that may contain an additional active agent or a subject in need thereof. It can be used for therapeutic purposes in any of the resulting combination formulations.

Thus, a further aspect of the invention is described above, preferably for use as an adjuvant in a therapeutic method for delaying the progression of natural or pathological skeletal muscle tissue loss and / or damage and / or disability. It is to provide a defined 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant.

More specifically, the present invention relates to an agent intended to delay the progression of natural or pathological skeletal muscle tissue loss and / or damage and / or disorder in a subject in need thereof. Concerning the use of the 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulants described herein.

In other words, the present invention is a therapeutic method for delaying the progression of natural or pathological skeletal muscle tissue loss and / or damage and / or disorder in a subject in need thereof, in an effective amount of the book. The present invention relates to a method comprising the step of administering to the subject the 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant described herein.

In yet another aspect, the present invention
i) Promotes self-renewal and / or differentiation of satellite cells; and / or ii) With respect to pharmaceutical compositions for use as agents that prevent and / or inhibit the depletion of satellite cell pools.
The composition comprises at least one of the above defined 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulants and at least one pharmaceutically acceptable excipient.

More specifically, the present invention is described above for the production of agents for i) promoting self-renewal and / or differentiation of satellite cells and / or ii) preventing and / or inhibiting depletion of satellite cell pools. Regarding the use of pharmaceutical compositions.

In other words, the present invention is a method of i) promoting self-renewal and / or differentiation of satellite cells and / or ii) preventing and / or inhibiting depletion of a satellite cell pool, wherein an effective amount of the composition. The present invention relates to a method comprising a step of administering to a subject in need thereof.

"Pharmaceutically acceptable excipients" as used herein are of pharmaceutical grades that improve the delivery, stability or bioavailability of an activator and are metabolizable and non-toxic by the subject to whom it is administered. Means a compound. Preferred excipients according to the invention include any excipient commonly used in pharmaceutical formulations such as microcrystalline cellulose, lactose, starch, and soybean powder.

According to a preferred embodiment, the pharmaceutical composition delays, prevents, or treats the loss and / or damage and / or disorder of natural or pathological skeletal muscle tissue (5-HT1 BR according to the invention). (Although it may have different efficacy than stimulants) and / or enhances or enhances the action of 5-HT1 BR stimulants (ie, 5-hydroxytryptamine 1B receptors exerted by 5-HT1 BR stimulants) It further comprises at least one activator (which increases or enhances the stimulation of body activity). The agent can be, for example, a therapeutic agent known to delay, prevent, or treat the loss and / or damage and / or disorder of natural or pathological skeletal muscle tissue.

Such activators are well known and, but not limited to, satellite cells, mesenchymal stem cells, hematopoietic stem cells, pericutaneous cells, mesodermal hemangioblasts (the cells described above are native or inherited). , Anti-inflammatory agents such as corticosteroids and NSAIDs (non-steroidal anti-inflammatory agents), muscle nutritional agents such as myostatin, immunotherapeutic agents, antibodies, genetic elements such as CRISPR / Cas9, and them. Combinations, preferably satellite cells, genetically modified satellite cells, mesenchymal stem cells, hematopoietic stem cells, pericutaneous cells, mesenchymal hemangioblasts and combinations thereof. Pindolol and (S)-(-)-pindolol are also suitable activators as they are known to enhance the action of antidepressants. In fact, the above-mentioned active agent combination formulation with the 5-HT1 BR stimulant according to the present invention promotes self-regeneration and / or differentiation of satellite cells, and / or prevents and / or inhibits the depletion of the satellite cell pool. It can be significantly improved, thereby improving muscle regeneration.

For the purposes of the present invention, among the 5-HT1 BR stimulants and the above active agents, delaying, preventing or treating, and / or treating the loss and / or damage and / or damage of natural or pathological skeletal muscle tissue. It is within the skill of one of ordinary skill in the art to select the appropriate combination of activators that increase or enhance the action of the 5-HT1 BR stimulant.

The pharmaceutical composition of the present invention may preferably be in a form suitable for the object of the present invention. For example, the composition can be in a form suitable for parenteral, oral or topical administration, eg, a liquid suspension, a solid dosage form (granule, pill, capsule or tablet), or a paste or gel. The term parenteral, as used herein, includes subcutaneous, intravenous, or intramuscular injection. For example, the pharmaceutical composition may be in a form suitable for intramuscular administration.

More specifically, the composition can be used to slow the progression of natural or pathological skeletal muscle tissue loss and / or damage and / or damage.

Thus, in certain embodiments, the invention is defined above for use in therapeutic methods for slowing the progression of natural or pathological skeletal muscle tissue loss and / or damage and / or disability. Provide pharmaceutical compositions.

More specifically, the present invention is described above for producing a drug intended to delay the progression of natural or pathological skeletal muscle tissue loss and / or damage and / or disability in a subject in need thereof. Regarding the use of pharmaceutical compositions.

In other words, the invention is a therapeutic method for delaying the progression of natural or pathological skeletal muscle tissue loss and / or damage and / or disability in a subject in need thereof, wherein an effective amount of said. The present invention relates to a method comprising the step of administering a pharmaceutical composition to the subject.

As indicated above, the 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulants according to the invention delay, prevent, natural or pathological skeletal muscle tissue loss and / or damage and / or damage. It is particularly advantageous to combine or treat and / or combine with an active agent that enhances or enhances the action of 5-HT1 BR stimulants conventionally used in the art.

Thus, a further aspect of the invention is a 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant, either naturally or as a combination formulation for simultaneous, individual or sequential administration in a subject in need thereof. To provide an active agent that delays, prevents or treats the loss and / or damage and / or disorder of pathological skeletal muscle tissue and / or enhances or enhances the action of 5-HT1 BR stimulants. Is.

Preferred by the present invention to delay, prevent or treat the loss and / or damage and / or damage of natural or pathological skeletal muscle tissue and / or enhance or enhance the action of the 5-HT1 BR stimulant. The activator is as described above.

Dosage and administration schemes for 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulants or pharmaceutical compositions according to the invention will be more adapted to those of skill in the art depending on the age, body weight and severity of symptoms of the subject being treated. It is possible.

Thus, in another aspect, the 5-hydroxytryptamine 1B receptor (5-HT1 BR) or the composition according to the invention is once daily, preferably for about 6 weeks (particularly slow-acting 5-HT1 such as fluoxetine). It can be administered for about 12 days (especially for fast-acting 5-HT1 BR stimulants such as vortioxetine).

Further, in a preferred embodiment, the 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant or composition according to the invention is from about 5 mg / kg to about 30 mg / kg, preferably from about 10 mg / kg to about 25 mg / kg. It is administered at a dose of 18 mg / kg, comprising between kg, more preferably between about 15 mg / kg and about 20 mg / kg.

In another embodiment, the 5-hydroxytryptamine 1B receptor stimulant according to the invention can be used for drug screening purposes. In particular, novel drug assays may be provided that identify therapeutic agents that effectively interfere with the self-renewal and / or differentiation of satellite cells and / or the replenishment of in vivo satellite cell pools.

In this aspect, the invention is more particularly in vitro for identifying agents or combinations of agents that promote self-renewal and / or differentiation of satellite cells and / or prevent and / or inhibit the depletion of satellite cell pools. It ’s a screening method.
a) The step of contacting the isolated satellite cells with a candidate drug or a combination of candidate drugs;
b) Step of evaluating the cell phenotype of the cell;
c) The cell phenotype of step b) is contacted with the satellite cell phenotype in the absence of the drug or drug combination and / or the 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant as described above. The present invention relates to a method comprising a step of comparing with the phenotype of the satellite cells.

Preferably, the isolated satellite cell in step a) is a quiescent satellite cell.

The above method may optionally, based on the comparison of step c), determine whether the candidate agent or combination of agents promotes self-renewal and / or differentiation of satellite cells and / or prevents depletion of the satellite cell pool and /. Alternatively, the step d) for determining whether or not to inhibit may be further included.

Cell phenotype can be assessed by analyzing the expression of cell markers that are characteristic of satellite cells during quiescence, self-renewal and / or differentiation as described above.

The present invention is better understood in the light of the detailed description of the following experiments, including examples. However, one of ordinary skill in the art will recognize that this detailed description is not limiting and that various modifications, substitutions, omissions, and modifications can be made without departing from the scope of the invention.
The present invention is as follows.
[1] i) A self-renewal and / or differentiation promoter of satellite cells; and / or
ii) Drugs that prevent and / or inhibit the depletion of satellite cell pools
5-Hydroxytryptamine 1B receptor (5-HT1 BR) stimulant for use as a stimulant.
[2] The agent for use according to [1] above, wherein the agent is used in a subject affected by natural or pathological loss and / or damage and / or disorder of skeletal muscle tissue.
[3] The agent for use according to [1] or [2] above, wherein the natural loss and / or damage and / or disorder of the skeletal muscle tissue is sarcopenia.
[4] The agents are antidepressants and antimitral agents, their pharmaceutically acceptable derivatives, analogs, isomers, metabolites, salts, solvates, inclusions, polymorphs, and co-crystals, and The agent for use according to any one of the above [1] to [3], which is selected from the group consisting of a combination thereof.
[5] The antidepressants are atypical antidepressants, preferably bisarylsulfanylamines such as vorthioxetine, and thianeptin, agomelatin, nefazodon, trazodon, buspirone, tandospirone, and ketamine; selective serotonin reuptake inhibitors (SSRIs),. Preferably, fluoxetin, citalopram, escitapram, sertralin, norceltraline, paroxetin, fluboxamine, femoxetin, indalpine, alaprocrate, sericlamine, ifoxetin, dimeridine, dapoxetin, etoperidone, and desmethylcitalopram, didesmethylcyta, etc. Metabolites of Serotonin and Norepinephrine Reuptake Inhibitors (SNRIs), preferably duroxetine, benrafaxin, desbenrafaxin, mirunashiplan, levomirunashiplan, and citalopram; serotonin-norepinefurin-dopamine reuptake inhibitors (SNDRI), preferably bisifazine, brasofensin, tesofensin, and nomifencin; tricyclic antidepressants (TCA), preferably clomipramine, amoxapine, nortryptiline, maprotirin, trimipramine, imipramine, decipramine and protryptiline; monoamine oxidase. Inhibitors (MAOs), preferably iproniazide, phenergine, tranylicipromin, moclovemid, selegiline and rasagirin; and noradrenalinergic / specific serotoninergic antidepressants (NaSSA), preferably miltazapine, myanserin, aptazapine, esmiltazapine. , Setiptyrin and S32212 (also known as N- [4-methoxy-3- (4-methylpiperazin-1-yl) phenyl] -1,2-dihydro-3H-benzo [e] indol-3-carboxyamide). The agent for use according to [4] above, which is selected from the group consisting of.
[6] The anti-mitiginal pain agent is ergotamine or triptan, and the triptan is preferably from sumatriptan, lizatriptan, solmitriptan, eletriptan, almotriptan, flovatriptan, naratriptan, abitriptan and donitriptan. The agent for use according to [4] above, which is selected from the group consisting of.
[7] The use according to any one of [1] to [6] above, wherein the agent is modified to contain at least one charged chemical moiety, preferably positively charged. Drug for.
[8] The agent for use according to [7] above, wherein the positively charged chemical moiety is a quaternary ammonium group or a tertiary sulfonium group.
[9] -The quaternary ammonium group is of formula (I).
^- (NR ₁ R ₂ R ₃ ) ⁺ Z- (I)
[During the ceremony,
Z is an organic or inorganic anion; and
R ₁ , R ₂ and R ₃ are independently selected from the group consisting of alkyl, aryl and cycloalkyl, respectively].
Do you have;
-The tertiary sulfonium group is of formula (II).
^- (SR ₄ R ₅ ) ⁺ Z- (II)
[During the ceremony,
Z is an organic or inorganic anion; and
R ₄ and R ₅ are independently selected from the group consisting of alkyl, aryl and cycloalkyl, respectively]
The agent for use according to the above [8].
[10] The agent is a salt of vortioxetine, histidine, arginine or ricine, pyrrolidinium-vortioxetine, piperazinium-vortioxetine, dimethylammonine-vortioxetine, sulfonium-vortioxetine, N-oxide-vortioxetine, sulfoxide-vortioxetine, and phosphonium-vortioxetine. The agent for use according to any one of the above [7] to [9], which is a positively charged vortioxetine selected from the group consisting of vortioxetine coupled to a charged amino acid.
[11] The agent for use according to the above [10], wherein the agent is histidine-vortioxetine or pyrrolidinium-vortioxetine.
[12] Defined in any of the above [1]-[11] for use in therapeutic methods for slowing the progression of natural or pathological skeletal muscle tissue loss and / or damage and / or disability. The drug to be done.
[13] i) A promoter of self-renewal and / or differentiation of satellite cells; and / or
ii) Drugs that prevent and / or inhibit the depletion of satellite cell pools
A pharmaceutical composition for use as a
Includes at least one 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant as defined in any of [1]-[11] above and at least one pharmaceutically acceptable excipient. The composition.
[14] Delay, prevent or treat the natural or pathological loss and / or damage and / or damage of skeletal muscle tissue and / or increase or enhance the action of the 5-HT1 BR stimulant. The composition for use according to [13] above, further comprising at least one activator.
[15] The drug is a satellite cell, a hematopoietic stem cell, a pericyte cell, a hemangioblast, a mesenchymal stem cell, an anti-inflammatory drug, a muscle nutrient, an immunotherapeutic drug, an antibody, a genetic element, a pindrol, (S)-(-). -The composition for use according to [14] above, selected from the group consisting of pindrils and combinations thereof.
[16] The 5-hydroxytryptamine 1B receptor (5-HT1) defined in any of the above [1] to [11] as a combination preparation for simultaneous, individual or sequential administration to a subject in need thereof. BR) stimulants and delay, prevent or treat the natural or pathological loss and / or damage and / or damage of skeletal muscle tissue and / or increase the action of the 5-HT1 BR stimulants. Or an activator that enhances.
[17] In vitro 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant defined in any of the above [1] to [11] for promoting self-renewal and / or differentiation of satellite cells. use.
[18] A method for promoting self-renewal and / or differentiation of satellite cells in vitro, defined in any of the above [1] to [11] in an effective amount in an isolated biological sample containing satellite cells. A method comprising the step of administering a 5-hydroxytryptamine 1B receptor (5-HT1 BR) stimulant.
[19] An in vitro screening method for identifying agents or combinations of agents that promote self-renewal and / or differentiation of satellite cells; and / or prevent and / or inhibit the depletion of satellite cell pools.
a) The step of contacting the isolated satellite cells with a candidate drug or a combination of candidate drugs;
b) Step of evaluating the cell phenotype of the cell;
c) The cell phenotype of step b) is defined as the phenotype of satellite cells in the absence of the agent or combination of agents and / or 5-hydroxytryptamine as defined in any of the above [1]-[11]. Steps to compare with the phenotype of satellite cells contacted with 1B receptor (5-HT1 BR) stimulants
A method that includes.

Key genetic markers expressed in adult skeletal muscle satellite cells (MSCs) and progenitor cells (MPCs) (extracted from Shi et al., 2006). Fluoxetine increases the number of blood vessels and satellite cells (SC) in skeletal muscle. (A) Schematic of fluoxetine delivery and time of sacrifice. (B) Number of satellite cells per mm ² in section. (C-d) Tissue sections of placebo-treated mouse tibialis anterior muscle (c) and fluoxetine-treated TgPax7nGFP mouse (d). Arrows point to satellite cells. (E) Number of blood vessels per mm ² after intraperitoneal (IP) or oral administration of fluoxetine at various time points. (Fg) Placebo (f) and Fluoxetine Treatment (g) Tissue sections of tibialis anterior muscle of Flk1 ^{GFP / +} mice. The picture shows endogenous GFP. (H) Number of CD31 + cells in subcutaneously grafted Matrigel plugs of placebo and fluoxetine treated mice. (IJ) Representative images of Matrigel plugs after 6 weeks in C57Bl / 6 mice treated with placebo (i) or fluoxetine (j). (K-l) Representative images of blood vessels (arrows) within the Matrigel plug detected by HE staining in placebo (k) or fluoxetine (l) treated mice. (M) Vessel length 4 days after seeding of cytodex® beads covered with HUVEC. (N) Representative images of cytodex® beads 4 days after sowing in the presence of placebo plasma. (O) Representative images of cytodex® beads 4 days after seeding in the presence of fluoxetine-treated mouse plasma. Use of immunostaining and FACS cell sorting in FIGS. (P)-(v): (p) Number of Pax7GFP-expressing cells per digested TA as counted by FACS (expressed as absolute number). (Q) Representative FACS profile of digested TA of TgPax7nGFP mice. (R) BrdU + SC. Demanded BrdU with fluoxetine or placebo in drinking water from start to end of treatment. Tg: Cumulative number of Pax7nGFP mice (n = 3 at each time point). TA muscle was digested, cells were isolated by FACS, rotated on slide glasses and immunostained against BrdU. (S) Tg: Percentage of BrdU + cells in Pax7nGFP mice (n = 3 at each time point). Tg: Pax7nGFP mice were treated with fluoxetine orally and injected twice with BrdU (12 and 4 hours prior to death). TA muscle was digested, cells were isolated by FACS, rotated on slide glasses and immunostained against BrdU. (T) The number of blood vessels per mm ² counted on tissue sections using the CD31 immunolabel in placebo and fluoxetine treated animals. (Uv) Representative tissue sections of laminin and CD31 + immunostaining in placebo (u) and fluoxetine (v) treated mice. Regarding FIGS. (A) to (o): n = 8 mice were used for each condition except for in vivo test cells of n = 6. The data are average ± s. d. It is represented by. ^* P <0.05; ^** P <0.01; ^*** P <0.001. The scale bar represents 100 μm. Regarding FIGS. (P) to (v): n = 7 mice were used for each condition except for the BrdU test at each time point n = 3. The data are average ± s. d. Represented by. ^** P <0.01. The scale bar represents 100 μm. Fluoxetine improves muscle regeneration by increasing the number of satellite cells. (A) Schematic of fluoxetine delivery, muscle injury, BrdU injection and time of sacrifice. (B) Number of Pax7GFP + cells per mm ² 4 days after injury. (C-d) Immunostaining of Pax7GFP + cells to sections after treatment with placebo (c) and fluoxetine (d). (E) Number of (myogenin +) cells during differentiation 4 days after injury. (Fg) Representative photographs of myogenin and GFP cells in placebo (f) and fluoxetine (g) treated animals. (H-i) Hematoxylin and eosin staining of frozen sections TA 14 days after injury in placebo (h) and fluoxetine (i) treated animals. (J) Fiber size μm ² in placebo and fluoxetine 14 days after injury. (K) Percentage of fibrotic area 14 days after injury in placebo and fluoxetine treated mice. (L) Hematoxylin and eosin staining of frozen sections TA after a series of injuries. Use n = 7 mice for each condition, except for counting n = 9 GFP + cells. The data are average ± s. d. It is represented by. ^* P <0.05; ^** P <0.01; ^*** P <0.001. The scale bar represents 100 μm. Muscle regeneration is faster after fluoxetine treatment and the number of self-regenerating satellite cells is higher. (A) Number of immune Gr1 (granulocytes) and F4 / 80 (macrophages) in placebo and fluoxetine treated mice 4 and 14 days after injury. (B-c) Tissue sections (fibrosis) of Sirius red staining 14 days after injury in placebo (b) and fluoxetine (c) treated animals. (D) Placebo vs. fluoxetine Calcium deposition 14 days after injury in treated animals. (E) Number of fibers in placebo vs. fluoxetine treated animals 14 days after injury. (F) Number of blood vessels in Flk1 ^{GFP / +} mice 28 days after injury in placebo vs. fluoxetine treated animals. (G) Number of SCs in TgPax7nGFP mice 28 days after injury in placebo vs. fluoxetine treated animals. (H) Number of SCs after several injuries in placebo vs. fluoxetine treated animals. (I) The first split evaluated by an in vitro live video microscope. Cells were seeded with plasma from either placebo-treated C57Bl / 6 animals or fluoxetine-treated C57Bl / 6 animals. (J) Fission rate as assessed by an in vitro live video microscope. Cells were seeded with plasma from either placebo-treated C57Bl / 6 animals or fluoxetine-treated C57Bl / 6 animals. (K) Percentage of (myogenin +) cells during differentiation 4 days after seeding. Cells were seeded with plasma from either placebo-treated C57Bl / 6 animals or fluoxetine-treated C57Bl / 6 animals. Use n = 7 mice for each condition. The data are average ± s. d. It is represented by. ^* P <0.05; ^** P <0.01; ^*** P <0.001. The scale bar represents 100 μm. The effect of fluoxetine on endothelial and satellite cells is achieved via the 5-HT1 B receptor. (A) Scheme for delivery of fluoxetine and inhibitors. (B) RT-qPCR quantification of various serotonin receptors shown as an increase factor for placebo in endothelial and satellite cells. (C) Number of differentiated cells (myogenin +) per mm ² in placebo, fluoxetine and fluoxetine + GR127935 5-HT1 B antagonist 4 days after injury. (D) Fiber size μm ² 14 days after injury in placebo, fluoxetine and fluoxetine + GR127935 5-HT1 B antagonist. (E) Percentage of fibrotic area 14 days after injury. (F) The number of SCs per TA of Tg: Pax7nGFP mice in fluoxetine, fluoxetine and the inhibitor GR127935, the inhibitor GR127935 alone. (G-h) Representative photographs of the number of GFP + cells on sections in fluoxetine (g) and fluoxetine and GR127935 5-HT1 B inhibitor (h). This picture shows endogenous GFP. (I) Number of blood vessels in Flk1 ^{GFP / +} mice after fluoxetine treatment and GR127935 5-HT1 B antagonist. Representative tissue sections of the number of blood vessels in fluoxetine (j), fluoxetine and inhibitor (k) per (j-k) mm ² (counted using endogenous GFP from Flk1 ^{GFP / +} mice). N = 7 mice were used for each condition (control n = 5). The data are average ± s. d. Expressed as. ^* P <0.05; ^** P <0.01; ^*** P <0.001. The scale bar represents 100 μm. 5-HT1 BR is involved in the fluoxetine effect, but 5-HT2 BR is not. (A) Number of SCs 4 days after injury in placebo, fluoxetine, fluoxetine and GR127935 5-HT1 BR antagonist and MDL100907 5-HT2 BR antagonist. (B) Calcium deposition 14 days after injury in placebo, fluoxetine, fluoxetine and GR127935 5-HT1 BR antagonists. (C) Number of immune Gr1 (granulocytes) and F4 / 80 (macrophages) 4 days after injury in placebo, fluoxetine, fluoxetine and GR127935 5-HT1 B antagonist and MDL100907 5-HT2 BR antagonist. (D) Number of immune Gr1 (granulocytes) and F4 / 80 (macrophages) 14 days after injury in placebo, fluoxetine, fluoxetine and GR127935 5-HT1 BR antagonist and MDL100907 5-HT2 BR antagonist. (E) Number of differentiated cells (myogenin +) 4 days after injury when fluoxetine and fluoxetine and 5-HT2 BR antagonists are used. (F) Number of SCs from TgPax7nGFP after 5-HT2 BR inhibition by MDL100907 antagonist. (G) Number of blood vessels from Flk1 ^{GFP / +} after 5-HT2 BR inhibition by MDL100907 antagonist. N = 6 mice were used for each condition (control n = 5). The data are average ± s. d. Expressed as. ^* P <0.05; ^** P <0.01; ^*** P <0.001. The scale bar represents 100 μm. In vitro plasma from fluoxetine-treated mice accelerates differentiation in both mouse and human satellite cells at an early stage and increases self-renewal at a later stage. (A) Scheme of in vitro fluoxetine and inhibitor delivery. (B) Time-dependent percentage of Pax7 + cells in the total number of FACS-selected SCs in in vitro placebo plasma, fluoxetine plasma, fluoxetine plasma and GR127935 inhibitors, fluoxetine plasma and MDL100907 inhibitors. (C) Self-regenerating (also called reserve cells) SC (Pax7 + / EdU-) 14 days after seeding of FACS-selected SC in in vitro placebo plasma, fluoxetine plasma, fluoxetine plasma and GR127935 inhibitor, fluoxetine plasma and MDL100907 inhibitor. Number of. (D) Time-dependent percentage of MyoD + cells in the total number of FACS-selected SCs in in vitro placebo plasma, fluoxetine plasma, fluoxetine plasma and GR127935 inhibitors, fluoxetine plasma and MDL100907 inhibitors. (E) Time-dependent percentage of myogenin + cells in the total number of FACS-selected SCs in placebo plasma, fluoxetine plasma, fluoxetine plasma and GR127935 inhibitors, fluoxetine plasma and MDL100907 inhibitors in vitro. (F) First cell division assessed by live video microscopy in in vitro placebo plasma, fluoxetine plasma, fluoxetine plasma and GR127935 inhibitors, fluoxetine plasma and MDL100907 inhibitors. (G) Division rates assessed by live video microscopy in in vitro placebo plasma, fluoxetine plasma, fluoxetine plasma and GR127935 inhibitors, fluoxetine plasma and MDL100907 inhibitors. (H) Percentage of Pax7 + cells co-cultured in vitro with CP94253 5HT1B specific agonists confirmed by immunofluorescence. (I) Percentage of myogenin + cells co-cultured in vitro with CP94253 5HT1BR specific agonists confirmed by immunofluorescence. (J) Differentiated (myogenin +) cells derived from primary human SC obtained from pre-seeding technique 4 days after seeding with placebo plasma, fluoxetine plasma, fluoxetine plasma and GR127935 inhibitor, fluoxetine plasma and MDL100907 inhibitor in vitro. .. (K) Self-renewal (Pax7 + / EdU) derived from primary human SC obtained from pre-seeding technique 4 days after seeding with placebo plasma, fluoxetine plasma, fluoxetine plasma and GR127935 inhibitor, fluoxetine plasma and MDL100907 inhibitor in vitro. -)cell. Except for human myoblasts with n = 4 for each condition, n = 6 mice were used for each condition. The data are average ± s. d. It is represented by. ^* P <0.05; ^** P <0.01; ^*** P <0.001. The scale bar represents 100 μm. Fluoxetine improves the phenotype of dystrophy mice. (A) Quantification of necrotic area (mm ² ) in placebo and fluoxetine treated animals. (B) Fiber size (area) of placebo and fluoxetine treated Mdx mice. (C-d) Hematoxylin and eosin staining of frozen sections TA of Mdx mice treated with either placebo (c) or fluoxetine (d). (E) Number of blood vessels in Mdx mice treated with either placebo or fluoxetine, counted by immunostaining with CD31 and expressed as the number of cells per mm ² . (F) Number of cells (Pax7 + and BrdU + cells) during the cycle in Mdx mice treated with either placebo or fluoxetine. (G) Luminex® experiment on interleukin 6 (IL6) in picograms / gram of protein expression levels in plasma of mice treated with both placebo and fluoxetine. (H) Luminex® experiment on interleukin 10 (IL10) representing picogram / gram protein expression levels in plasma of mice treated with both placebo and fluoxetine. (I) Number of Gr1 + cells in frozen section TA of placebo-treated and fluoxetine-treated Mdx mice. The number is shown as an absolute number per cross section. Antagonism to 5HT1B-R eliminates the beneficial effects of fluoxetine on the dystrophy phenotype. (A) Quantification of necrotic area (mm ² ) in placebo, fluoxetine and fluoxetine and GR127935 5HT1BR inhibitor treated animals. (B) Placebo, fluoxetine and fluoxetine and GR127935 5HT1BR inhibitor-treated Mdx mice fiber size (area). (C-e) Hematoxylin and eosin staining of frozen sections TA of Mdx mice treated with either placebo (c) or fluoxetine (d) or fluoxetine and GR127935 inhibitor (e). (F) Number of blood vessels in Mdx mice treated with placebo, fluoxetine or fluoxetine and a GR127935 5HT1BR inhibitor, counted by immunostaining with CD31 and expressed as the number of cells per mm ² . (G) Number of cells (Pax7 + and EdU + cells) during the cycle in Mdx treated with either placebo, fluoxetine or fluoxetine and a GR127935 5HT1BR inhibitor. (H) Number of Gr1 + cells in frozen section TA of placebo-treated and fluoxetine-treated Mdx mice. The number is shown as an absolute number per cross section. N = 9 mice for each condition. The data are average ± s. d. It is represented by. ^* P <0.05; ^*** P <0.001; ns: Not significant. The scale bar represents 100 μm. (I) Grip strength test. The total strength of animals was measured in fluoxetine and placebo-treated mice (limbs). (J) Maximum tension (6V) of edl muscle fibers in fluoxetine or placebo-treated mdx mice. (K) Tension of isolated edl at various voltages in placebo and fluoxetine treated mdx mice. Vortioxetine increases the number of blood vessels and satellite cells in vivo. (A) Schematic of the timing of vortioxetine delivery and sacrifice. (B) I. The number of blood vessels counted in the intercept after CD31 immunostaining per mm ² after P treatment. (C) P. The number of blood vessels counted in the intercept after CD31 immunostaining per mm ² after O treatment. (D) Placebo, 12 days and 3 weeks vortioxetine I. P-treated Tg: Number of satellite cells counted by FACS in Pax7nGFP mice. (E) Placebo, 12 days and 3 weeks vortioxetine P. et al. O-treated Tg: Number of satellite cells counted by FACS in Pax7nGFP mice. Vortioxetine increases the number of blood vessels and satellite cells in vivo and in vitro via 5-HT1 B receptors. (A) Vortioxetine I.V. in TgPax7nGFP mice at 20 mg / Kg for 12 days. The number of blood vessels per mm ² counted on the section by CD31 immunostaining after P treatment. (B) Vortioxetine I.T. of TgPax7nGFP at 20 mg / Kg for 12 days. Number of satellite cells per anterior tibial muscle counted by FACS after P treatment. (C-e) cells were sorted by FACS from Tg: Pax7nGFP mice and seeded at 2000 cells / cm ² . The next day, vortioxetine was added at 10 μM. At the indicated time points, cells were fixed and stained for Pax7 (c), Myod (d), MyoG (e). The vortioxetine derivatives histidine-vortioxetine and pyrrolidinium-vortioxetine increase the number of vascular and satellite cells: (a-b) cells were sorted by FACS from Tg: Pax7nGFP mice and seeded at 2000 cells / cm ² . The next day, histidine-vortioxetine or pyrrolidinium-vortioxetine was added at 10 μM. (A) Percentage of Pax7 + cells at various time points in vitro with PBS or vortioxetine or histidine-vortioxetine or pyrrolidinium-vortioxetine. (B) Percentage of MyoG + cells at various time points in vitro with PBS or vortioxetine or histidine-vortioxetine or pyrrolidinium-vortioxetine. (C-d) PBS (n = 4) or histidine-vortioxetine (n = 5) or pyrrolidinium-vortioxetine (n = 5) was injected IP into Tg: Pax7nGFP mice at 20 mg / kg for 12 days. Muscle (TA) was digested and counted by cytometry. (C) shows histidine-vortioxetine vs. PBS injection. (D) shows pyrrolidinium-vortioxetine vs. PBS. The data are average ± s. d. Expressed as. ^** P <0.01.

Fluoxetine, vortioxetine and their derivatives increase muscle stem cells and improve muscle regeneration. material and method
1.1. Injection and Injury in Mice All procedures in this study have been approved by the Animal Care and Use committee (CETEA 2014-004) of the Pasteur Institute. Unless otherwise indicated, age male mice were used in this study and were bred in a temperature and humidity controlled pathogen removal facility at a 12:12 light / dark cycle. Food and drinking water were given freely.

Animals were anesthetized with ketamine (Imalgene 1000 100 mg / Kg Military) and xylazine (Rompun 2% 20 mg / Kg Bayer) prior to injury. Animals were hydrated and treated with analgesics (buprenorphine axis 0.3 mg / kg) twice daily for 4 days after injury. For injury, mice were anesthetized as previously described and 10 μl of 12.5 μg / ml noteoxan was injected into the tibialis anterior muscle. All protocols were reviewed by the Pasteur Institute, the supervisory authority for compliance with French and European regulations under Animal Welfare Law and Public Health Service recommendations. This project has been reviewed and approved by the Pasteur Institute Ethics Board (C2EA 89-CETEA) (# 2013-0044).

Among treated mice, Flk1 ^{GFP / +} mice in which green fluorescent protein (GFP) is targeted at the VEGF-receptor-2 locus and exhibits a bright GFP signal to all endothelial cells are Alexander Medvinsky (Institute for Stem Cell Research). , Edinburgh University, Edinburgh, UK) courtesy of.

1.2. Histological analysis The tibialis anterior muscle (TA) was carefully dissected, snap frozen in liquid nitrogen-cooled isopentane for a few minutes, and stored at -80 ° C until frozen sectioning (10 μm section). Sections were maintained overnight at room temperature prior to staining. The sections were then rehydrated in PBS for 10 minutes and fixed in 10% formalin for 3 minutes. These sections were then routinely stained with hematoxylin and eosin (HE) using an automated stainer or manually using Sirius Red.

These glass slides were double-blind and automatically evaluated if possible (fiber diameter, total number of cells, infarcted area).

1.3. Immunostaining Immunostaining is performed by fixing with 4% paraformaldehyde (PFA EMS # 15710) in cold PBS, permeating with 0.5% Triton X-100 at room temperature for 20 minutes, washing and washing with 10% BSA. This was done for frozen sections that were blocked for 30 minutes. Sections were incubated overnight at 4 ° C. with the primary antibody (see Table 1 below) and for 45 minutes with the Alexa-bound secondary antibody 1/250 and Hoechst. The sections are then subjected to automatic axioscan (Zeiss) or inverted Observer. Analysis was performed using the Z1 Photome (Zeiss). For apoptosis assessment, cells were harvested in 2% serum, rotated on polyD-lysine (Sigma-Aldrich # P6407) and immediately fixed at PFA 4%.

1.4. Cell sorting, counting and incision of cultured muscle were performed in cold DMEM as previously described. The muscles were then chopped with small scissors and placed in a 50 ml falcon tube containing 0.1% collagenase and 0.25% trypsin while gently penetrating at 37 ° C. After 20 minutes, the supernatant was collected in 20% serum placed on ice and collagenase / trypsin solution was added to continue digestion. When the muscle was completely digested, the solution was filtered using a 40 μm cell strainer. Satellite cells were cultured in 1: 1 DMEM-Glutamax (Gibco # 41965-039): MCDB201 (Sigma # M6770) containing 20% serum FBS (Bioest S1860). The medium was filtered using a 0.22 μm filter. Cells were seeded on a Matrigel coating (BD Biosciences # 354234) and maintained in an incubator (37 ° C., 5% CO ₂ ). In some in vitro studies, plasma was extracted from fluoxetine or vortioxetine-treated animals by cardiac puncture after 6 weeks and then centrifuged at 1500 g for 15 minutes. The supernatant thus obtained was used as a substitute for FBS in the culture medium, and the remaining medium was unchanged.

For the counting of satellite cells, only the tibialis anterior muscle was cut out, digested as described above, and the entire tube was analyzed to evaluate the number of satellite cells per muscle. FACS analysis was performed using FACSasia (Beckman). All analyzes and quantifications were performed using Summit v4.3 software and FloJo software from DakoCytomation. To eliminate dead cells, cells were labeled with propidium iodide 10 μg / ml (Sigma-Aldrich # P4170) and labeled against the FACS profile using PE (phycoerythrin, red) channels.

1.5. Cells isolated by live video microscopy FACS were seeded overnight in a 24-well glass bottom plate (P24G-0-10-F; MatTek) coated with Matrigel (BD Biosciences # 354234) and pre-equilibrated in an incubator. The plate was placed in medium (1: 1 DMEM Glutamax: MCDB [Sigma-Aldrich], 20% FCS (Bioest S1860). The plate was then incubated at 37 ° C., 5% CO ₂ (Zesis, Pecon). A Zeiss Observer.Z1 operated with a PlnN 10 × / 0.8W phaseII objective lens and an AxioCam camera operated by AxioVision were used. Cells were imaged for up to 5 days with a brightfield and phase filter and 30 with a MozaiX 3X3 (Zesis). Images were obtained every minute. Raw data was converted and provided as a video.

1.6. Image Analysis For image analysis (fibrosis quantification), ImageJ 1.46r software was used for at least 3 sections for each test and 10 different photographs taken at random for each section. Pixel values were collected after converting these photographs to binary images. For fiber size, sections were immunostained overnight at 4 ° C. with rabbit anti-laminin (Sigma-Aldrich # L9393) diluted 1/200. A secondary donkey anti-rabbit 488 (DL488 Jackson Immuno # 711486152) was used at room temperature for 45 minutes at 1/200. Demarcation of fiber boundaries was done automatically by using Pixicavator® software.

1.7. Luminex® (Multiple Immunoassay)
Thaw a quick-frozen plasma sample (n = 6 for each condition) and extract the supernatant for Luminex® multiple cytokine and chemokine analysis (Bio-Plex® ProTM Mouse Cytokine Standard 23plex, Group I and Standard 9plex). , Group II). Normalization was performed by the sample weight of the frozen muscle.

1.8. RT-qPCR
Total RNA was isolated from cells using the RNAeasy Micro kit (Qiagen). Total RNA was reverse transcribed using Superscript® III Reverse Transcriptase (In vitro gen). Real-time quantitative PCR was performed using the Power System Green PCR Master Mix (Applied Biosystems) and the dye incorporation rate was monitored using the StepOne ™ Plus RealTime PCR system (Applied Biosystems). At least 3 biological iterations were used for each condition. Data were analyzed by StepOne Plus RT PCR software v2.1 and Microsoft Excel. GAPDH transcript levels were used for normalization of each target (= ΔCT). Real-time PCR CT values were analyzed using the 2- ( _ΔΔCt ) method and the expression factor was calculated (ΔΔCT method, Livak et al., 2001).

1.9. Force Measurement The grip strength test is a non-invasive method designed to assess mouse muscle strength in vivo. A grip meter (Bio-GT3, BIOSEB) attached to the force transducer measures the maximum force generated. Placebo and fluoxetine (6 weeks treatment) mdx mice were placed with their four paws grounded on the grid and gradually pulled backwards until the mice released their grips. Statistical analysis was performed after 5 tests and mean values were calculated using 3 median data for each mouse. Results are expressed as the result of three maximum forces (g) normalized to body weight (g).

1.10. Exfoliated fiber test TA muscle was excised from placebo and fluoxetine treated mice (6 weeks treatment). A bundle of 2-5 fibers was manually isolated from the muscle as previously described. Chemical stripping was performed using Triton X-100. The detached fibers were attached to a Displacement Measurement System KD 2300 (Model 0.5SU, Kaman Instrumentation, Colorado Springs, CO, USA). To measure force, the exfoliated fiber preparation was incubated in a laxative (pCa 9.0, low calcium content) containing 1% Triton X-100 (v / v) for 1 hour to incubate the sarcoplasmic reticulum and muscle. The sarcoplasmic reticulum membrane was solubilized and then washed several times in a detergent-free laxative solution. The fibers were adjusted to a slack length and then gradually stretched to maximum tension. Isometric tensions were continuously recorded using a tut recorder (Model 1200, Linear, Reno, Nevada, USA). The tension obtained was normalized to the fiber cross-sectional area.

1.11. Statistical analysis Statistical analysis was performed using GraphPad Prism software with appropriate tests (nonparametric Mann-Whitney, if not specified) and a confidence interval of at least 95% for significance; p. The values are <0.05 (*), <0.01 (**), and <0.001 (***). The figure shows the mean ± SD for all test animals or ± SEM for RT-qPCR, or as shown.

2. 2. result
2.1. Fluoxetine 2.1.1. Fluoxetine increases the number of blood vessels in skeletal muscle and the number of satellite cells. To examine the effect of fluoxetine on the number of blood vessels, 18 mg / kg of fluoxetine intraperitoneally (IP) or orally in Flk1 ^{GFP / +} or TgPax7nGFP mice. When (PO) administration was performed for 3 or 6 weeks (Fig. 2a), direct visualization of endothelial cells and satellite cells (SC) was possible, respectively. This study focused on the tibialis anterior muscle (TA). In skeletal muscle, SC is the center of muscle repair. Histological counts showed that the number of SCs was higher when treated with fluoxetine (9.7 ± 3.1 SC / mm ² with placebo vs. 16.3 ± 5.4 SC with treatment, p = 0.04) (FIGS. 2b to d and p to q). These results were confirmed by digesting TA and directly counting the number of SCs by cytometry using Tg: Pax7nGFP mice (Sambasivan et al., 2009) (p = 0.02) (Fig. 2p, q). SCs are located close to capillaries and angiogenesis is known to be important for the survival of muscle repair SCs with cellular interactions between vascular cells and SCs. Therefore, the number of blood vessels was quantified. In control Flk1 ^{GFP / +} mice (placebo), the number of blood vessels was 870 ± 213.1 blood vessels / mm ² , and I.I. It increased after P treatment (1103 ± 110.2 vessels / mm ² ) and further increased at 6 weeks (1384 ± 175.3 vessels / mm ² , p = 0.008) (FIG. 2e). The new vessels exhibited a normal histological appearance connecting the basement membrane with the permeable lumen (which may contain red blood cells). Since inflammation and neoangiogenesis are correlated in some models (Sadat et al., 2014), daily fluoxetine I. et al. P administration could cause unwanted peritonitis that could interfere with angiogenesis. Therefore, fluoxetine was used for 6 weeks, P.I. When O was administered, a similar increase was observed in the number of blood vessels (1256 ± 62.2 blood vessels / mm ² p = 0.008, FIGS. 2e to g). These results showed that fluoxetine was applied to C57Bl / 6 mice for 6 weeks. It was also confirmed in another model by O administration and immunolabeling of CD31 + cells to frozen section TA. In the placebo-treated mice, 841 ± 137 CD31 + cells / mm ² were counted against 2260 ± 361 CD31 + cells / mm ² in the fluoxetine-treated mice, and p = 0.008 (FIGS. 2t to v), confirming the previous findings. After 6 weeks in vivo delivery of fluoxetine, IL-1a, IL-1b, IL-2, and eotaxin levels were down-regulated, while IL-4 and IL-13 were up-regulated (p = 0). .02). Other test cytokines showed no significant changes (Table 2).

To further confirm the above results, an ex vivo Matrigel angiogenesis assay was used. To do this, cold Matrigel was introduced subcutaneously, which solidified, penetrated by host cells and p. Allows the formation of new blood vessels in O-treated C57Bl / 6. The number of CD31-expressing cells was higher with the fluoxetine-treated plug (72.9 ± 22.6 CD31 + cells / mm ² ) than with placebo (25.20 ± 13.8 CD31 + cells / mm ² , p = 0.002). (Figs. 2h to l), the previous findings were confirmed.

These data were further confirmed by in vitro HUVEC assay (human endothelial cells). C57Bl / 6 mice were used for 6 weeks on P. O treatment was performed and plasma was extracted from the blood. HUVEC cells were then seeded on cytodex beads and cultured in either plasma. Four days after sowing, HUVECs incubated with plasma from treated animals grew faster (p≤0.0001) (FIGS. 2m-o).

Cell division was quantified during total oral treatment with fluoxetine and after BrdU administration, and their results showed that 90% of the SC population was dividing (FIG. 2r). A short pulse of BrdU 16 hours before death showed that the majority of SCs were dividing between the 3rd and 5th weeks (Fig. 2s).

2.1.2. Fluoxetine Improves Muscle Regeneration Brain In order to investigate whether fluoxetine had a functional effect on muscle, fluoxetine was treated with notexin damage (Fig. 3a) and Tg: muscle regeneration ability in Pax7nGFP mice. Was examined (Sambasivan et al., 2009). Comparison of muscles 4 and 14 days after injury showed better regeneration in both cases (FIGS. 3b-j). In fact, there was a significant difference in muscle regeneration characteristics between placebo and fluoxetine-treated mice. Four days after injury, treated animals showed more SC (179 ± 283 at treatment vs. 177.2 ± 49 at placebo, p = 0.04) (FIGS. 3b-d) and more cells. Was already being differentiated (p = 0.0006) (FIGS. 3e-g). In the placebo group 14 days after injury, the regenerating central nucleus fibers were of varying size (large and small, 81 ± 28.4 μm ² ), with multiple endomysial infiltrations by mononuclear inflammatory cells (9.14 per section). ± 3.9 Gr1 + cells and 12.6 ± 3.9 F4 / 80 + cells), the presence of multiple large basal foci of calcium deposition (19 ± 4 / mm ² ), and mild endomysial fibrosis. (6.4 ± 2% of the total muscle area) was shown (FIGS. 3h to k). In contrast, in fluoxetine-treated mice, the regenerating fibers are larger, less variable in size (129 ± 12.6 μm ² , p = 0.005), and less inflammatory cells (4.1 ± 2.6 Gr1 + cells). P = 0.018 and 4.7 ± 2.7 F4 / 80 cells; p = 0.0017), low calcium deposition (3.2 ± 3 / mm ² p = 0.0006), and low endomysium Fibrosis (total muscle area 2.2 ± 0.7 p = 0.007) was shown (FIGS. 3h-k and 4a-d). However, the number of fibers remained the same (p = 0.4, FIG. 4e). Twenty-eight days after injury, the muscles were completely regenerated and there was a difference between these two groups, except that the fluoxetine-treated animals had higher vessel counts (p = 0.036) and SC counts (p = 0.076). I couldn't see it. (Fig. 4f, g). These data were confirmed in vitro by seeding SC from Tg: Pax7nGFP mice in 20% plasma from C57Bl / 6 treated mice or placebo. By live video microscopy, the first satellite cell division was faster (26.45 hours ± 1.18 after seeding for fluoxetine-treated plasma, 29.08 hours ± 0.37 after seeding for control plasma, p = 0.02). It was found to occur at a high rate of division (12.5 ± 2.1 hours with placebo vs. 8.7 ± 0.7 hours with fluoxetine; p = 0.047) (Fig. 4i, j). .. In addition, 4 days after seeding, the number of myogenin-positive cells was higher (12 ± 5.5% of total seeded cells in placebo, 34.75 ± 6.3% in fluoxetine treatment; p = 0.02) (Fig.). 4k) and faster myogen formation were also seen, confirming faster differentiation and repair of myogens in vivo. Interestingly, along with this improvement in regeneration, there was also a reduction in post-injury muscle inflammation as before (Table 3). For example, the pro-inflammatory cytokine IL-6 decreased from 4258 ± 665 pg / μl of injury to 2459 ± 920 pg / μl of injury with fluoxetine treatment (p = 0.02).

Multiple injuries were applied to the fluoxetine treatment to further stimulate the muscles with the aim of confirming that the satellite cell (SC) pool was not depleted by deteriorating phenotype. The number of SCs per TA was maintained even after 3 injuries (Fig. 4h). At the tissue level, muscle regenerated well both injured and re-injured (Fig. 3l), indicating that SC was still a functional stem cell after fluoxetine treatment.

2.1.3. The effect of fluoxetine on blood vessels and satellite cells is obtained through stimulation of 5-HT1 B serotonin receptors. After treatment with O-fluoxetine, endothelial cells (CD34 +, CD31 +, Sca-1 +, CD45-) from digested muscle were subjected to cell sorting by FACS (FIG. 5a). RT-qPCR was then performed on serotonin receptor subtypes to further characterize which can activate endothelial cells (FIG. 5b). A 90 ± 35-fold increase in 5-HT1 BR subtypes in treated mice compared to placebo mice (p = 0.0035) and 30 ± 13 (p = 0) in 5-HT2 BR subtypes in fluoxetine-treated animals. A 5-fold increase was seen (Fig. 5b). Other test subtypes (5-HT1 AR, 5-HT1 DR, 5-HT1 FR, 5-HT2 AR, 5-HT2 CR) did not show a statistically significant increase (FIG. 5b). These data are available on P.M. for 6 weeks. Confirmed using endothelial markers from digested muscle after O-fluoxetine treatment (data not shown). The same findings were obtained with SC cells sorted by FACS from Tg: Pax7nGFP mice (FIG. 5b).

To investigate the role of 5-HT1 BR, GR127935 hydrochloride inhibitor (5-HT1 BR antagonist) was added to fluoxetine P. et al. For 6 weeks. It was delivered to an osmotic pump with O treatment, and blood vessel counting in Flk1 ^{GFP / +} mice and SC counting in Tg: Pax7nGFP mice were performed. The number of blood vessels was lower in fluoxetine and inhibitor-treated Flk1 ^{GFP / +} mice (1028 ± 173 blood vessels) compared to fluoxetine and PBS-treated Flk1 ^{GFP / +} mice (1949 ± 576 blood vessels / mm ² , p = 0.0159). / Mm ² ) (Figs. 5i-k). The number of SCs was also lower in fluoxetine and inhibitor-treated Tg: Pax7nGFP mice (4674 ± 1414 SC per TA) compared to fluoxetine and PBS-treated Tg: Pax7nGFP mice (7283 ± 2325SC p = 0.04 per TA). 5f-h). A 30-fold increase in 5-HT2 B receptor was detected after fluoxetine treatment, but counteracting it with MDL100907 (5-HT2 BR antagonist) did not show any inhibition of the fluoxetine effect (Fig. 6f, g).

After injury, inhibition of 5-HT1 BR by GR127935 decreased the number of SC (FIG. 6a) and differentiated cells (FIG. 5c) 14 days after injury, reduced fiber size (FIG. 5d) and increased calcium deposition (FIG. 5d). The beneficial effects of fluoxetine treatment were derived with increased fibrosis percentage (FIG. 5e) and increased infiltration of immune Gr1 and F4 / 80 cells (FIGS. 6c, d). After injury, inhibition of 5-HT2 BR by MDL100907 did not suppress the beneficial effects of fluoxetine (FIG. 6e).

These results were confirmed in vitro (Fig. 7a). The number of Pax7-positive cells decreased more rapidly after 4 days of seeding in plasma from fluoxetine-treated animals (24% ± 6 with fluoxetine treatment vs. 39.25% ± 8 p = 0.02 with placebo) ( FIG. 7b) was close to that of placebo when incubated with GR127935 (35.25% ± 6.2 p = 0.32) (FIG. 7b). 14 days after seeding, more reserve cells (in vitro) in fluoxetine-treated animal-derived plasma (9.4% ± 0.7) than in placebo-treated animals (4.4% ± 0.7, p = 0.01). (Static Pax7 positive cells) were detected, and when GR127935 was added in vitro, the number of reserve cells decreased (4.5% ± 0.5, p = 0.9) (FIGS. 7b, c). .. MyoD (activation marker) expression was always different when examined under three test conditions (FIG. 7d), whereas the differentiation marker myogenin (MyoG) was seeded with plasma from fluoxetine-treated mice. It showed a 3-fold increase in expression (p = 0.0017) (FIG. 7e). This difference disappeared when SC was sown with GR127935 (p = 0.4) (FIG. 7e). The presence of faster quiescence and higher split rate also disappeared with the addition of GR127935 in vitro (Fig. 7f, g). These effects of plasma from fluoxetine-treated animals persisted with the addition of MDL100907 (FIGS. 7b-g). Importantly, in vitro, direct addition of 5-HT1B agonist to culture medium elicited the same effect as addition of plasma from fluoxetine-treated mice (Fig. 7h, i). We found faster differentiation in the early post-sowing time and higher self-renewal rate in the late post-sowing time.

Similar results were seen when primary human myoblasts were seeded in plasma from fluoxetine-treated mice, with faster differentiation (p = 0.05) 4 days after seeding (Fig. 7j) and higher self-renewal of cells after seeding 14. Was obtained (p = 0.02) (Fig. 7k). These effects disappeared when 5-HT1 BR was canceled, but not when 5-HT2 BR was canceled (Fig. 7j, k).

2.1.4. Fluoxetine Improves Mdx Phenotype Fluoxetine was applied to Mdx mice (Bulfield G et al., 1983), a Duchenne muscular dystrophy mouse model, for 6 weeks. O. Delivered. Fluoxetine-treated Mdx mice showed fewer necrotic foci (mean 2893 ± 803 mm ² ) compared to untreated Mdx controls (mean 5041 ± 1629 mm ² , p = 0.04) (FIG. 8a). The fiber size was also generally larger in Mdx treated animals (3,192 ± 0.3 p = 0.051 for placebo vs. 4.241 ± 0.9 pixels for treatment) (FIG. 8b). Therefore, the number of regenerated foci was reduced in Mdx mice treated with fluoxetine (FIGS. 8c-d). The number of vessels increased in treated mice (1459 ± 327 vessels / mm ² ) compared to placebo (942.4 ± 113 vessels per section, p = 0.008) (FIG. 8e). Interestingly, the number of satellite cells during the cycle was also reduced, correlating with the previously seen reduction in the number of regenerated foci (Fig. 8f). Treated mice also showed lower cytokine levels compared to placebo (580 ± 158 pg / ml IL6, p = 0.004) (34.52 ± 21 pg / ml IL6, Table 4) (FIG. 8 g, Table 4). ). The same reduction was seen for other pro-inflammatory cytokines (data not shown). Note that IL10 (anti-inflammatory cytokine) levels were unchanged in treated animals (99.6 ± 56 pg / ml IL10) compared to placebo (64.1 ± 50, p = 0.4). I want to (Fig. 8h, Table 4). This finding was consistent with the reduced infiltration of inflammatory cells seen in muscle (9.6 ± 3.7 Gr1 + cells per section in placebo vs. 4 ± 2 in treated mice. .4 Gr1 + cells p = 0.04) (Fig. 8i). In particular, when Mdx mice were treated with fluoxetine and 5-HT1B inhibitors, the beneficial effects disappeared (FIGS. 9a-h).

At the functional level, total muscle strength in fluoxetine-treated mdx mice was increased by 56% (Fig. 9i), and isolated single fibers of extensor digitorum longus (edl) force were also increased by 45% (Fig. 9j). ~ K). This was confirmed by soleus muscle (data not shown).

2.2. Vortioxetine 2.2.1. Vortioxetine increases the number of vessels in the tibialis anterior muscle To examine the effect of vortioxetine on the number of vessels, C57Bl / 6 mice were given 20 mg / kg intraperitoneal (IP) of vortioxetine for 12 days, 3 weeks or 6 It was done in one of the weeks (Fig. 10a). The number of CD31 + cells was counted by immunostaining. The number of blood vessels in the placebo was (863.5 ± 115.6 blood vessels / mm ² ), and I.I. Increased after P treatment (2274 ± 926 vessels / mm ² , p = 0.03) and further increased after 3 weeks (2847 ± 705 vessels / mm ² , p = 0.02) (FIG. 10b). However, there was no statistically significant difference between the 12-day and 3-week treatments (p = 0.49). The new vessels exhibited a normal histological appearance connecting the basement membrane and the permeable lumen (which may contain red blood cells). Daily IP administration of vortioxetine could cause unwanted peritonitis that could interfere with angiogenesis, as inflammation and neoangiogenesis are correlated in some models. Therefore, when vortioxetine was orally administered (PO) for 12 days, a similar increase was observed in the number of blood vessels (2451 ± 595 blood vessels / mm ² p = 0.028, FIG. 10c). To further confirm these results, vortioxetine was added to Flk1 ^{GFP / +} mice for 12 days. O and I. P-administered plasma from ex vivo Matrigel angiogenesis assay and vorthioxetine-treated mice is used against HUVEC (endothelial cells).

2.2.2. Vortioxetine increases satellite cell counts Tibialis anterior muscle (TA) of Tg: Pax7nGFP mice was digested and analyzed by FACS in placebo and vortioxetine (12 days, 20 mg / Kg) treated mice to count satellite cell numbers. .. Next, when the cells per TA were counted by cytometry (FIG. 10d), the number of SCs was increased in the treated mice (8660 satellite cells ± 699) as opposed to the untreated mice (4444 satellite cells ± 802 p = 0.008). A clear increase was seen (Fig. 10a). When these data were confirmed by histological counting on the sections, a 2-fold increase was seen with respect to the TA sections. After 3 weeks of vortioxetine treatment, the number of satellite cells was much higher (9351 satellite cells ± 706, p = 0.0079), but there was no statistically significant difference between the 12 days and 3 weeks of treatment. (P = 0.15). Vortioxetine is also available at 20 mg / kg. When O administration was also performed, a similar increase in the number of SCs was observed, and as a result, I. The P result was confirmed (Fig. 10d, e).

2.2.3. Vortioxetine increases the number of blood vessels and satellite cells through stimulation of 5-HT1 B receptors To investigate the role of 5-HT1 BR, a specific 5-HT1 BR antagonist GR127935 hydrochloride inhibitor 12 Daily vortioxetine I. Delivered to the osmotic pump with P treatment. The number of blood vessels was lower in vortioxetine and inhibitor-treated Flk1 ^{GFP / +} mice (928 ± 207) compared to vortioxetine and PBS-treated Flk1 ^{GFP / +} mice (2274 ± 926 blood vessels / mm ² , p = 0.028). Blood vessel / mm ² ) (Fig. 11a). The number of SCs was also lower in vortioxetine and inhibitor-treated Tg: Pax7nGFP mice compared to vortioxetine alone (5540 ± 411 SCs per TA) (FIG. 11b). When it was counteracted by the 5-HT2 B receptor inhibitor MDL100907, no inhibition of the effect of vortioxetine was observed in either the number of blood vessels or the number of SCs (FIGS. 11a, 11a).

After injury, inhibition of 5-HT1 BR by GR127935 suppressed the beneficial effects of vortioxetine 12-day treatment and, in fact, reduced the number of SC and differentiated cells 14 days after injury, decreased fiber size and calcium deposition. An increase, an increase in the percentage of fibrosis and an increase in infiltration of immune Gr1 and F4 / 80 cells could be observed.

These results were confirmed in vitro as shown in FIGS. 11c-e. Satellite cells were isolated from Tg: Pax7nGFP mice and the cells were seeded at 2.000 cells / cm ² . After one night, vortioxetine was added at 10 μM. The number of Pax7-positive cells decreased more rapidly after 4 days of seeding (23% ± 2 in vortioxetine treatment vs. 38.25% ± 6 p = 0.02 in PBS) (FIG. 11c) with GR127935 inhibitor. It was close to the control (PBS) (25.5% ± 4.2 p = 0.32) when incubated (Fig. 11c). 14 days after seeding, vorthioxetine-treated cells (9% ± 0.7) had more reserve cells (Pax7-positive cells quiescent in vitro) than PBS (3.5% ± 0.9, p = 0.01). When detected and the GR127935 inhibitor was added in vitro, the number of reserve cells was reduced (3.5% ± 0.9, p = 0.9) (FIG. 11c). The expression of MyoD (activation marker) was not always different when examined under the three test conditions (Fig. 11d). However, the differentiation marker myogenin (MyoG) showed a 2-fold increase in expression 4 days after seeding with vortioxetine (p = 0.0017) (FIG. 11e). This difference in expression disappeared when SC was seeded with GR127935 5-HT1 BR inhibitor (p = 0.4) (FIGS. 11c-e). This positive effect of vortioxetine disappeared when GR127935 was added with vortioxetine in vitro (FIGS. 11c-e). However, these effects persisted when 5-HT2 BR antagonists were added (FIGS. 11c-e). This means that the positive effects seen when vortioxetine is seeded with satellite cells in vitro are mediated by 5HT1B receptors but not by 5HT2B.

These in vitro studies clearly show that vortioxetine acts directly on satellite cells and stimulates their differentiation at a faster regeneration rate than fluoxetine, similar to the findings obtained in vivo. Most importantly, an increase in the number of satellite cells during self-renewal was seen 14 days after in vitro, which underscores the ability of vortioxetine to increase the muscle stem cell pool.

2.3. Vortioxetine derivative 2.3.1. Histidine-vortioxetine and pyrrolidinium-vortioxetine increase the number of vascular and satellite cells Two derivatives of vortioxetine, namely histidine-vortioxetine and pyrrolidinium-vortioxetine, have equivalent or improved effects compared to unmodified vortioxetine. To determine if this is possible, the in vitro approach described in 22.3 above was used to the satellite cell differentiation cascade, Pax7 (hepatocyte) described in 22.3 above. It was examined by evaluating the expression patterns of sex and quiescent markers) and myogenin (MyoG, a marker of differentiation). To do this, satellite cells from Tg: Pax7-nGFP were isolated by FACS and seeded at 2.000 cells / cm ² (overnight seeding). The next day, vortioxetine, a derivative thereof, or control PBS was added at 10 μM and cells were fixed at the indicated time points.

The rate of cell differentiation was faster with vortioxetine, pyrrolidinium-vortioxetine and histidine-vortioxetine than with PBS (control). In fact, 38.2 ± 6.7% of the cells were still Pax7 + in PBS after 4 days, whereas 23.7 ± 2.78% (p) when the cells were seeded with vortioxetine. ≤0.001), 25.25 ± 4% when sown with pyrrolidinium-vortioxetine (p ≤0.001), 22.5 ± 3.3% when sown with histidine-vortioxetine. (P ≦ 0.0001) (FIG. 12a). Furthermore, in the myogenin staining data, MyoG + cells 13.25 ± 2% in the PBS control, 34.5 ± 2.5% (p ≦ 0.0001) for vortioxetine, and 19.75 ± 0.47% for pyrrolidinium-vortioxetine. This finding was confirmed with (p ≦ 0.05) and 30 ± 1.7% (p ≦ 0.0001) for histidine-vortioxetine (FIG. 12b).

Taken together, these results indicate that pyrrolidinium-vortioxetine and histidine-vortioxetine induce rapid differentiation of satellite cells in vitro, similar to vortioxetine.

Importantly, when assessing the self-renewal of satellite cells, it was found that both pyrrolidinium-vortioxetine and histidine-vortioxetine showed more Pax7 + cells 14 days after seeding (medium change every 4 days). .. In fact, at the end of the in vitro differentiation process, 2 ± 0.4% of cells were Pax7 + with PBS, compared with 10.5 ± 0.65% with vortioxetine and with pyrrolidinium-vortioxetine. It was 12.5 ± 1.9% (p ≦ 0.01) and 12.25 ± 1.3% (p ≦ 0.05) for histidine-vortioxetine. This indicates that the number of cells undergoing self-renewal in vitro is higher with pyrrolidinium-vortioxetine and histidine-vortioxetine than with vortioxetine alone (FIGS. 12a, b; 20% increase). When 20 mg / Kg of histidine-vortioxetine or pyrrolidinium-vortioxetine was injected into TgPax7nGFP mice by IP injection for 12 days, the number of SCs doubled in TA. These results show that these two derivatives have comparable effects compared to vortioxetine (FIGS. 12c, d).

3. 3. Consideration
Selective serotonin reuptake inhibitors (SSRIs) antidepressants (eg, fluoxetine) have been routinely used to treat widespread mood disorders (Marsella et al., 1975). Interestingly, the use of fluoxetine to improve the regeneration of organs other than the brain has never been evaluated.

The results show that administration of vortioxetine (a well-known atypical antidepressant) as well as fluoxetine increased the number of blood vessels, and most importantly, satellite cells, in skeletal muscle. These results were confirmed using different approaches (genetic and immunostaining), routes of administration (intraperitoneal and oral) and different concentrations. These data were further confirmed ex vivo with a large number of blood vessels invading the Matrigel plug in fluoxetine-treated mice. In vitro, human endothelial cells HUVEC showed more division when seeded in plasma of fluoxetine-treated mice than when plasma derived from placebo-treated mice was used. Primary human satellite cells also showed differentiation at early seeding and higher self-renewal potential at late seeding.

Definitively, fluoxetine or vortioxetine-treated animals showed faster regeneration upon injection, which was sustainable with multiple injuries. Faster regeneration was assessed at both the genetic level (myogenin expression) and the histological level. Moreover, regeneration was faster with vortioxetine compared to fluoxetine (2 weeks vs. 6 weeks). In pursuit of a mechanism of action that may be involved in this enhancement of regeneration, the rate of activation is involved, as MyoD was detected at the same time point in treated and control mice both in vivo and in vitro. I didn't think it was there. In fact, the number of early satellite cells almost doubled in vivo. In vitro, satellite cells showed more cell division and faster differentiation when seeded in fluoxetine-treated mouse plasma.

When the self-regeneration of satellite cells was evaluated, it was further confirmed that both pyrrolidinium-vortioxetine and histidine-vortioxetine showed better effects than vortioxetine alone 14 days after seeding in vitro. Early after sowing (4 days), both pyrrolidinium-vortioxetine and histidine-vortioxetine were found to show faster differentiation.

It is also shown that it evoked quiescent escape of fluoxetine or vortioxetine satellite cells. In fact, more cells were detected after 6 weeks of treatment, which were BrdU +, which means that these surplus cells are derived from existing cell division (self-renewal). This can be the basis for faster muscle regeneration after injury. In fact, higher satellite cell numbers may explain faster differentiation, but are more likely to explain their faster activation and faster division rate during in vitro injury. This can mean that satellite cells are quiescent, but escape from their dormant state and are rapidly activated when injured or in need of activation.

Thus, the in vivo and in vitro data presented herein show a two-step effect that can have different mechanisms depending on the physiological or pathological state of the cell. During dormancy, satellite cells can be activated by fluoxetine or vortioxetine or 5-HT1B stimulation that elicit their self-renewal in a controlled and limited manner. At some point in the differentiation cascade after cell activation, 5-HT1BR activation can elicit faster differentiation (high cell density, or as shown, direct 5-HT1B receptor). For any reason that activation induces differentiation (not an exclusive hypothesis). Thus, 5-HT1BR stimulation induces dormant escape of satellite cells, increasing cell division rate, differentiation as needed as well as self-renewal under normal physiological conditions.

Delivery of fluoxetine also increased serotonin receptor 5-HT1 BR levels 90-fold in endothelial and SC cells, and drug delivery of 5-HT1 BR inhibitors for these increases in both vascular and satellite cell numbers. The effect of fluoxetine was counteracted. In vitro, fluoxetine increased the rate of differentiation and the number of reserved cells, but the addition of inhibitors counteracted their effects. This is important because it shows that fluoxetine or metabolites found in plasma produced in vivo by fluoxetine administration can act directly on SC by targeting 5-HT1 B receptors. It is a point.

Inhibition of 5-HT2 BR does not affect these results, which means that this effect of fluoxetine from plasma-treated mice, or directly mediated by vortioxetine, acts specifically on 5-HT1 B receptors. Show that.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and is an X-linked recessive progressive muscle wasting disease caused by a deficiency of the functional dystrophin protein (Emery, 2002). Structural defects found in DMD result in muscle cells that are more sensitive to mechanical stress, accompanied by significant ischemia, which results in muscle cell damage, continuous muscle fibrosis and regeneration, Induces decreased calcium homeostasis, chronic inflammatory response, fibrosis, and muscle necrosis. In individuals with DMD, these processes inevitably result in decreased gait shortly after the first 10 years, with death and shortened lifespan in 30 or 40 years due to cardiac-respiratory abnormalities. No cure for DMD is known, and although the causative gene was identified over 20 years ago, therapeutic studies have produced few clinically relevant results. Because of these characteristics, targeting both blood vessels (ischemia) and satellite cells (muscle regeneration) can be a major concern.

After delivery of fluoxetine to dystrophy mice (Mdx), large fluctuations in muscle fiber size and central nuclear fibers were still seen, but the number of necrotic foci decreased dramatically and the overall diameter of the fibers increased. became. The Luminex® assay also showed a reduction in total levels of cytokines, a well-known read-out of inflammation in Mdx mice. This was associated with a decrease in the number of infiltrated inflammatory cells.

Taken together, these results show that delivery of fluoxetine, vortioxetine, or their derivatives to dystrophy patients promotes dystrophy by increasing muscle regeneration, more specifically, the number of muscle stem cells (in addition to blood vessels). Show that it has the potential to mitigate. The data show that this effect is directly mediated by 5-HT1 B receptors expressed on both endothelial and muscle stem cells.

References

Claims (16)

Containing a direct stimulant for 5-hydroxytryptamine 1B receptor (5-HT1 BR),
i) Promoter of self-renewal and / or differentiation of satellite cells; or ii) Preventive and / or inhibitor of satellite cell pool depletion.
The agent, wherein the direct stimulant is vortioxetine or vortioxetine modified to include at least one charged chemical moiety. The agent according to claim 1, wherein the agent is used in a subject affected by natural or pathological loss and / or damage and / or disorder of skeletal muscle tissue. The agent according to claim 2, wherein the natural loss and / or damage and / or disorder of the skeletal muscle tissue is sarcopenia. The agent according to any one of claims 1 to 3, wherein the modified vortioxetine is vortioxetine modified so as to contain at least one positively charged chemical moiety. The agent according to claim 4, wherein the positively charged chemical moiety is a quaternary ammonium group or a tertiary sulfonium group. -The quaternary ammonium group is the formula (I).
-(NR ₁ R ₂ R ₃ ) ^{+ Z-} (I)
[During the ceremony,
Z is an organic or inorganic anion; and R ₁ , R ₂ and R ₃ are independently selected from the group consisting of alkyl, aryl and cycloalkyl, respectively].
Or-the tertiary sulfonium group is of formula (II)
-(SR ₄ R ₅ ) ^{+ Z-} (II)
[During the ceremony,
Z is an organic or inorganic anion; and _R4 and _R5 are independently selected from the group consisting of alkyl, aryl and cycloalkyl, respectively].
The agent according to claim 5. Vortioxetine, pyrrolidinium-vortioxetine, piperazinium-vortioxetine, wherein vortioxetine modified to contain at least one positively charged chemical moiety is coupled to a positively charged amino acid such as a salt of vortioxetine, histidine, arginine or lysine. The agent according to any one of claims 4 to 6, selected from the group consisting of dimethylammonium-vortioxetine, sulfonium-vortioxetine, N-oxide-vortioxetine, sulfoxide-vortioxetine, and phosphonium-vortioxetine. The agent of claim 7, wherein the vortioxetine modified to include at least one positively charged chemical moiety is histidine-vortioxetine or pyrrolidinium-vortioxetine. The agent as defined in any one of claims 1-8 for use in a therapeutic method for slowing the progression of natural or pathological skeletal muscle tissue loss and / or injury and / or disorder. i) A pharmaceutical composition for use as a promoter of self-renewal and / or differentiation of satellite cells; and / or ii) a preventive and / or inhibitor of depletion of satellite cell pools.
A direct stimulant of at least one 5-hydroxytryptamine 1B receptor (5-HT1 BR) as defined in any one of claims 1-8 and at least one pharmaceutically acceptable excipient. A composition comprising. Delays, prevents or treats the natural or pathological loss and / or damage and / or damage of skeletal muscle tissue and / or enhances or enhances the action of the direct stimulant of 5-HT1 BR. The composition according to claim 10, further comprising at least one activator. The active agents are satellite cells, hematopoietic stem cells, pericutaneous cells, hemangioblasts, mesenchymal stem cells, anti-inflammatory agents, myotrophic agents, immunotherapeutic agents, antibodies, genetic elements, pindrol, (S)-(-)-pindrol. , And the composition of claim 11, selected from the group consisting of combinations thereof. i) Promote self-renewal and / or differentiation of satellite cells; and / or
ii) Prevention and / or inhibition of satellite cell pool depletion
It is a combination product for use in
Direct stimulants of 5-hydroxytryptamine 1B receptor (5-HT1 BR) as defined in any one of claims 1-8 and the natural or pathological loss and / or damage of skeletal muscle tissue and / Or an activator that delays, prevents or treats the disorder, and / or enhances or enhances the action of the 5-HT1 BR stimulant , simultaneously, individually or to subjects in need thereof. A combination formulation for sequential administration . In vitro use of a direct stimulant of 5-hydroxytryptamine 1B receptor (5-HT1 BR) as defined in any one of claims 1-8 to promote self-renewal and / or differentiation of satellite cells. .. A method for in vitro promoting self-renewal and / or differentiation of satellite cells, wherein an effective amount of 5-hydroxytryptamine as defined in any of claims 1 to 8 is given to an isolated biological sample comprising satellite cells. A method comprising the step of administering a direct stimulant of a 1B receptor (5-HT1 BR). An in vitro screening method for identifying agents or combinations of agents that promote self-renewal and / or differentiation of satellite cells; and / or prevent and / or inhibit the depletion of satellite cell pools.
a) The step of contacting the isolated satellite cells with a candidate agent or a combination of candidate agents;
b) Step of evaluating the cell phenotype of the cell;
c) A satellite cell in which the cell phenotype of step b) is contacted with a direct stimulant of 5-hydroxytryptamine 1B receptor (5-HT1 BR) as defined in any one of claims 1-8. A method comprising a step of comparing with a phenotype.

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